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.

Angiotensin II stimulates phospholipases C and A2 in cultured rat mesangial cells

1987 ◽  
Vol 253 (1) ◽  
pp. C113-C120 ◽  
Author(s):  
D. Schlondorff ◽  
S. DeCandido ◽  
J. A. Satriano
Keyword(s):  
Arachidonic Acid ◽  
Angiotensin Ii ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Mesangial Cells ◽  
Inositol Trisphosphate ◽  
Pge2 Production ◽  
Diacylglycerol Lipase ◽  
Stimulation Of ◽  
In Cells

Angiotensin II stimulates prostaglandin (PG) E2 formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE2 are important in modulating glomerular function. We examined the mechanism for stimulation of PGE2 production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca2+-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE2 production in response to either angiotensin II or A23187. In contrast, TFP (50 microM) inhibited basal PGE2 production and stimulation by angiotensin II and A23187. TFP also decreased 14C release in response to angiotensin from cells prelabeled with [14C]arachidonic acid, which was associated with inhibition of 14C loss from phosphatidylinositol. In cells prelabeled with 32P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphospate. TFP enhanced 32P labeling of phosphatidylinositides, but did not prevent the loss of phosphatidylinositol 4,5-bisphosphate in response to angiotensin. This was verified in cells prelabeled with myo-[3H]inositol where angiotensin stimulated formation of [3H]inositol trisphosphate. TFP enhanced formation of [3H]inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in [32P]lysophospholipids, indicating activation of also phospholipase A2. This process was inhibited by TFP. Taken together, these results are consistent with stimulation of both phospholipase C and A2 by angiotensin, the latter step responsible for the release of arachidonic acid and PGE2 formation. The activation of phospholipase A2, but not that of phospholipase C, is inhibited by TFP, perhaps by interference with calmodulin-dependent steps.

scholarly journals The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes

1986 ◽  
Vol 238 (2) ◽  
pp. 425-436 ◽  
Author(s):  
C J Meade ◽  
G A Turner ◽  
P E Bateman
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cytochalasin B ◽  
Polymorphonuclear Leucocyte ◽  
Limiting Factor ◽  
Polymorphonuclear Leucocytes ◽  
Hydrolysis Of

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a ‘second signal’ (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.

scholarly journals Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP

1984 ◽  
Vol 222 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S E Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Free Arachidonic Acid ◽  
Stimulation Of

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.

scholarly journals The immediate phase of c-kit ligand stimulation of mouse bone marrow-derived mast cells elicits rapid leukotriene C4 generation through posttranslational activation of cytosolic phospholipase A2 and 5-lipoxygenase.

1995 ◽  
Vol 182 (1) ◽  
pp. 197-206 ◽  
Author(s):  
M Murakami ◽  
K F Austen ◽  
J P Arm
Keyword(s):  
Bone Marrow ◽  
Arachidonic Acid ◽  
Mast Cells ◽  
Cell Membrane ◽  
Phospholipase A2 ◽  
Mouse Bone Marrow ◽  
Kit Ligand ◽  
Cytosolic Phospholipase ◽  
Delayed Phase ◽  
Stimulation Of

c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.

Phospholipases in human parturition

1990 ◽  
Vol 2 (5) ◽  
pp. 511 ◽  
Author(s):  
T Wilson
Keyword(s):  
Protein Kinase C ◽  
Phospholipase A2 ◽  
Main Role ◽  
Kinase C ◽  
Arachidonate Release ◽  
Synthesis Regulation ◽  
External Calcium ◽  
Human Parturition ◽  
Stimulation Of

Phospholipase A2 plays a major role in controlling PG synthesis. Regulation of the activity of this enzyme probably holds the key to the onset of labour. Both PLA2 and PLC can contribute to arachidonate release and PG production in cells, but PLA2 appears to be the main role of synthesis. Phospholipase A2 and PLC can be activated independently of each other; an influx of external calcium is required for PLA2 activation. It is suggested that PLC contributes to PG synthesis through product stimulation of protein kinase C which maintains a pool of free arachidonate by inhibiting reincorporation into the cell membrane. The regulatory role for lipocortin in phospholipase inhibition is controversial and unlikely to be relevant to the onset of labour.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

2001 ◽  
Vol 85 (05) ◽  
pp. 882-889 ◽  
Author(s):  
Leslie Lockhart ◽  
Caroline Pampolina ◽  
Brent Nickolaychuk ◽  
Archibald McNicol
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Cytosolic Phospholipase A2 ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Low Concentrations ◽  
Acid Release ◽  
Induced Aggregation

SummaryThe release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

A role for p38 MAP kinase in platelet activation by von Willebrand Factor

2004 ◽  
Vol 91 (01) ◽  
pp. 102-110 ◽  
Author(s):  
Ilaria Canobbio ◽  
Stefania Reineri ◽  
Fabiola Sinigaglia ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Map Kinase ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
P38 Map Kinase ◽  
Cyclooxygenase Inhibitors ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Stimulation Of

SummaryPlatelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FcγRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin αIIbβ3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FcγRIIA by a specific monoclonal antibody or the prevention of thromboxane A2 synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. However, phosphorylation of p38MAPK was prevented by the tyrosine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWFstimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found thatVWF induced phosphorylation of cytosolic phospholipase A2, and that this process was prevented by the p38MAPK inhibitor SB203580.These results demonstrate that p38MAPK is a key element in the FcγRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A2 and arachidonic acid release.

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