Possible Pathways Of Arachidonic Acid Liberation As Studied With Purified Phospholipases

1981 ◽  
Author(s):  
H Chap ◽  
B Perret ◽  
G Mauco ◽  
M Plantavid ◽  
F Laffont ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Phospholipase C ◽  
Ph Dependence ◽  
Plasma Lipoproteins ◽  
Heterogeneous Distribution ◽  
Outer Leaflet ◽  
Membrane Bound

Two kinds of informations about arachidonic acid (AA) metabolism in platelet phospholipids (PL) have been obtained from the use of purified phospholipases: 1) Beside the determination of PL sidedness in the plasma membrane, non-lytic degradation by phospholipase A2 + sphingomyelinase C showed that only 6 % of the total platelet AA is localized in the outer surface of the plasma membrane. This heterogeneous distribution is actually a consequence of PL asymmetry, since sphingomyelin and phosphatidylcholine, which predominate in membrane outer leaflet, contain only traces or relatively lower amounts, respectively, of AA than the internal lipids. It is further shown that incubating platelets with free AA specifically labels the large internal pool of AA, whereas the small external pool is renewed by a direct exchange of phosphatidylcholine with plasma lipoproteins. This offers a doublelabelling method allowing to explore the exact role of each AA pool.2) Platelet aggregation by Clostridium welchii phospholipase C produces the same metabolic changes (accumulation of phosphatidic and lysophosphatidic acids) as those induced by thrombin. These observations have led to describe the existence of a cytosolic phosphatidylinositol-specific phospholipase C and a membrane-bound diglyceride lipase. Both enzymes, coupled to diglyceride− (and monoglyceride−) kinase(s), could achieve AA release and (lyso) phosphatidic acid accumulation. Some properties of these enzymes (subcellular localization, calcium and pH dependence, positional specificity) will be presented.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

scholarly journals Mechanisms of amphipath-induced stomatocytosis in human erythrocytes

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 782-786 ◽  
Author(s):  
SL Schrier ◽  
A Zachowski ◽  
PF Devaux
Keyword(s):  
Blood Cells ◽  
Shape Change ◽  
Drug Induced ◽  
Inhibiting Effect ◽  
Outer Leaflet ◽  
Human Red Blood Cells ◽  
Spin Resonance ◽  
Vanadate Inhibition

Abstract We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin- labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration- dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.

scholarly journals Levels of Human Erythrocyte Membrane-Bound and Cytosolic Glycohydrolases Are Associated with Oxidative Stress in Erectile Dysfunction Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
L. Massaccesi ◽  
G. V. Melzi d’Eril ◽  
G. M. Colpi ◽  
G. Tettamanti ◽  
G. Goi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Erectile Dysfunction ◽  
Plasma Membranes ◽  
Lag Time ◽  
Specific Marker ◽  
Human Erythrocyte Membrane ◽  
Key Enzyme ◽  
Membrane Bound

Oxidative stress (OS) and production of NO, by endothelium nitric oxide synthetase (eNOS), are involved in the pathophysiology of erectile dysfunction (ED). Moreover, OS induces modifications of the physicochemical properties of erythrocyte (RBC) plasma membranes and of the enzyme content of the same membranes. Due to their role in signalling early membrane alterations in OS-related pathologies, several plasma membrane and cytosolic glycohydrolases of human RBC have been proposed as new markers of cellular OS. In RBC, NOS can be activated and deactivated by phosphorylation/glycosylation. In this regulatory mechanism O-β-N-AcetylGlucosaminidase is a key enzyme. Cellular levels of O-GlcNAcylated proteins are related to OS; consequently dysfunctional eNOS O-GlcNAcylation seems to have a crucial role in ED. To elucidate the possible association between RBC glycohydrolases and OS, plasma hydroperoxides and antioxidant total defenses (Lag-time), cytosolic O-β-N-AcetylGlucosaminidase, cytosolic and membrane Hexosaminidase, membraneβ-D-Glucuronidase, andα-D-Glucosidase have been studied in 39 ED patients and 30 controls. In ED subjects hydroperoxides and plasma membrane glycohydrolases activities are significantly increased whereas Lag-time values and cytosolic glycohydrolases activities are significantly decreased. These data confirm the strong OS status in ED patients, the role of the studied glycohydrolases as early OS biomarker and suggest their possible use as specific marker of ED patients, particularly in those undergoing nutritional/pharmacological antioxidant therapy.

Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Ute Bank ◽  
Anke Heimburg ◽  
Astrid Wohlfarth ◽  
Gudrun Koch ◽  
Karsten Nordhoff ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Low Molecular Weight ◽  
Immune Functions ◽  
Cellular Functions ◽  
Fluorogenic Substrates ◽  
Cellular Effects ◽  
Viable Cells ◽  
Substrate Conversion ◽  
Membrane Bound

Abstract The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)2-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO2)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

scholarly journals Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

2009 ◽  
Vol 184 (3) ◽  
pp. 451-462 ◽  
Author(s):  
Jian-Jiang Hao ◽  
Yin Liu ◽  
Michael Kruhlak ◽  
Karen E. Debell ◽  
Barbara L. Rellahan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Membrane Association ◽  
Erm Proteins ◽  
Cytoskeletal Remodeling ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Hydrolysis Of

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

scholarly journals Novel Evidence of Expression and Activity of Ecto-Phospholipase C γ1 in Human T Lymphocytes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3833-3840
Author(s):  
Sebastiano Miscia ◽  
Angela Di Baldassarre ◽  
Amelia Cataldi ◽  
Rosa Alba Rana ◽  
Valerio Di Valerio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Cell Types ◽  
T Lymphocyte Subsets ◽  
Membrane Flow ◽  
Inositol 1 ◽  
Outer Leaflet ◽  
Expression And Activity

Abstract Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCγ1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCγ1-positive (PLCγ1+) cells (∼37%) were CD8+ and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and activity, which were restored by treatment with an antiproliferative agent like natural interferon beta. Moreover, the different kinetics of formation of its hydrolytic products, inositol 1 phosphate and inositol 1:2 cyclic phosphate (Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a selective involvement of the two inositol phosphates in the mechanisms regulating the metabolism of particular T-lymphocyte subsets.

Platelet Activation : Role of Exogenous and Endogenous Phospholipases

1979 ◽  
Author(s):  
B. Perret ◽  
G. Mauco ◽  
M.F. Simon ◽  
H. Chap ◽  
L. Douste-Blazy
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Alternative Pathway ◽  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Platelet Response ◽  
Phospholipid Hydrolysis ◽  
Outer Leaflet

Phosoholipase A2 from bee venom induces aggregation of human platelets, provided that phospholipid hydrolysis is enabled by simultaneous incubation with sphingomyelinase C. Inhibition of the platelet response by indomethacin indicates that aggregation is due to arachidonic acid release. On another hand, this model allows to describe an asymmetrie distribution of arachidonic acid, whose only 6% is located in the outer leaflet of the plasma membrane.During platelet aggregation by phospholipase C, the diacylglycerol and its hydrolysis product 2-acyl-glycerol are phosphorylated into phosphatide and lysophosphatidic acids, respectively. As the same kinds of changes occur in the presence of thrombin, a unifying hypothesis for platelet activation is proposed, involving the stimulation of an endogenous phospholipase C, whose some properties will be reported (neutral optimal pH, Ca-requlrement, phosphatidylinositol specificity and cytosol-localization). This model can be related to the recent finding that phosphatide acid behaves as a calcium-ionophore (Gerrard, J.M. et al., Prostaglandins Med., 1978, 1, 387) and provides an alternative pathway for arachidonic acid mobilization.

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