PHOSPHOLIPASE A2 ACTIVATION BY A MECHANISM SEPARATE TO THAT RESPONSIBLE FOR PHOSPHOLIPASE C STIMULATION IN ALPHA-THROMBIN-STIMULATED HUMAN PLATELETS

1987 ◽  
Author(s):  
Michael F Crouch ◽  
Eduardo G Lapetina
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Control Mechanisms ◽  
Induced Aggregation ◽  
Stimulation Of

The ability of cell surface receptor occupation to increase the activity of phospholipase A2 has been thought to be due to the prior activation of phospholipase C and an increase in the intracellular Ca2+ concentration. However, recent evidence from our and other laboratories has suggested that this may not be the case, but rather stimulation of phospholipase A2 may be under the control of separate receptor-activated events. We have investigated this further by comparing the ability of prostacyclin (PGI2) and epinephrine to alter platelet responses to thrombin and examining the resulting phospholipase A2 activities.Alpha-thrombin stimulated aggregation of human platelets, the formation of inositol phosphates and phosphatidic acid, mobilizaton of Ca2+ from internal stores and Ca2+ influx, protein phosporylation (47 kDa and 20 kDa) and arachidonic acid (AA) release. Each of these responses was partially inhibited by prostacyclin (PGI2) except that of AA release, which was abolished. In combination with epinephrine and PGI2, alpha-thrombin-induced aggregation, phosphatidic acid formation and protein phosphorylation were restored, but the release of AA only reached 50% of its control value. Epinephrine alone had no effect on any of these responses, either in the presence or absence of PGI2. Thus, alpha-thrombin-induced activation of phospholipase A2 is more sensitive to the effects of PGI2 than is phospholipase C, and supports the possibility that there are distinct control mechanisms for receptor activation of these enzymes. We are presently examining the role of Gs in the inhibition by PGI2 of platelet phospholipase A2 and of Gi in the thrombin stimulation of this enzyme

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 Sodium fluoride mimics effects of both agonists and antagonists on intact human platelets by simultaneous modulation of phospholipase C and adenylate cyclase activity

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 859-866
Author(s):  
J Kienast ◽  
J Arnout ◽  
G Pfliegler ◽  
H Deckmyn ◽  
B Hoet ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Sodium Fluoride ◽  
Inositol Phosphates ◽  
Atp Release ◽  
Phosphodiesterase Inhibitor ◽  
Human Platelets ◽  
Camp Phosphodiesterase ◽  
Induced Aggregation ◽  
Camp Levels

Using intact human platelets, we studied the effect of sodium fluoride (NaF) on platelet aggregation and release reaction and correlated the functional changes to intracellular events specific for either agonist- induced or antagonist-induced platelet responses. At lower concentrations, with a peak activity between 30 and 40 mmol/L, NaF induced aggregation and release of adenosine 5′-triphosphate (ATP) that was associated with increased formation of inositol phosphates, a rise in cytosolic free Ca2+, and phosphorylation of 20-kd and 40-kd proteins. At NaF concentrations greater than 40 mmol/L, aggregation and ATP release decreased dose-dependently in parallel with a decrease in Ca2+ mobilization, whereas neither inositol phosphate formation nor 40- kd protein phosphorylation was reduced. At these concentrations, NaF caused a dose-dependent transient rise in platelet cyclic adenosine 3′,5′-monophosphate (cAMP) levels that was sufficient to account for the observed reduction in Ca2+ mobilization, aggregation, and ATP release. Stimulated cAMP levels started declining rapidly within 30 seconds of addition of NaF, however. Similarly, prostacyclin (PGI2)- induced cAMP accumulation was temporarily enhanced but subsequently suppressed by NaF, suggesting either stimulation of a cAMP phosphodiesterase or delayed inhibition of adenylate cyclase. Evidence for the latter was provided by the finding that NaF pretreatment of platelets resulted in partial inhibition of PGI2-stimulated cAMP formation in the presence of the cAMP phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (MIX). We conclude that NaF exerts a dual (stimulatory and inhibitory) effect on adenylate cyclase in intact platelets that is accompanied by simultaneous activation of a phosphoinositide-specific phospholipase C; in addition, a cAMP phosphodiesterase may be activated.

Microbubble-Induced Phospholipase C Activation Does not Correlate with Platelet Aggregation

1993 ◽  
Vol 69 (04) ◽  
pp. 394-396 ◽  
Author(s):  
R Malmgren ◽  
T Thorsen ◽  
A Nordvik ◽  
H Holmsen
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Human Platelets ◽  
Similar Extent ◽  
Arachidonic Acid Metabolites ◽  
Acid Metabolites ◽  
Rule Out ◽  
Stimulation Of

SummaryThe effect of nitrogen-(N2-)microbubbles on platelets resembles that of common platelet agonists with respect to aggregation and secretion, but is considerably slower and is poorly inhibited by aspirin. This paper reports the effect of microbubbles on platelet phospholipase C activity in gelfiltered human platelets prelabelled with [32P]Pi ([32P]-GFP). The experiments were run in the presence of an ADP scavenging system in order to rule out effects of ADP. Stimulation of [32P]-GFP for 30 min with microbubbles caused a significant reduction in single platelets (p <0.0004) and a significant increase in 32P-activity in the phosphatidic acid (PA) fraction (p <0.02). Epinephrine potentiated the microbubble-induced reduction in single platelets (p <0.05), but did not enhance the amount of 32P in the platelet [32P]PA fraction. The 32P-radioactivity in the PI-fraction increased with time to a similar extent when [32P]-GFP was stirred for 30 min in absence of microbubbles as it did after 30 min of agonist exposure. There were no significant changes in the [32P]PIP and [32P]PIP2 fractions. Aspirin abolished the microbubble-induced increase in 32P-activity in the PA fraction, but had no significant effect on the reduction in single platelets. Aspirin had a small but significant, reducing effect on platelet aggregation induced by a combination of epinephrine and microbubbles (p <0.05). With epinephrine, however, aspirin did not completely abolish the increase in [32P]-PA. It is concluded that microbubbles alone cause platelets to aggregate by a novel mechanism that operates independent of cyclooxygenase-dependent arachidonic acid metabolites and phospholipase C activation.

Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation

1996 ◽  
Vol 271 (3) ◽  
pp. C895-C904 ◽  
Author(s):  
S. Lajat ◽  
Z. Tanfin ◽  
G. Guillon ◽  
S. Harbon
Keyword(s):  
Phospholipase C ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Similar Increase ◽  
Inositol 1,4,5 Trisphosphate ◽  
M3 Subtype ◽  
Subtype A ◽  
Stimulation Of

The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.

Stimulation and binding of myocardial phospholipase C by phosphatidic acid

1995 ◽  
Vol 269 (2) ◽  
pp. C349-C358 ◽  
Author(s):  
R. A. Henry ◽  
S. Y. Boyce ◽  
T. Kurz ◽  
R. A. Wolf
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Ventricular Myocytes ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Adult Rabbit ◽  
High Affinity ◽  
Rabbit Ventricular Myocytes ◽  
Stimulation Of

Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

scholarly journals Sodium fluoride mimics effects of both agonists and antagonists on intact human platelets by simultaneous modulation of phospholipase C and adenylate cyclase activity

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 859-866 ◽  
Author(s):  
J Kienast ◽  
J Arnout ◽  
G Pfliegler ◽  
H Deckmyn ◽  
B Hoet ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Sodium Fluoride ◽  
Inositol Phosphates ◽  
Atp Release ◽  
Phosphodiesterase Inhibitor ◽  
Human Platelets ◽  
Camp Phosphodiesterase ◽  
Induced Aggregation ◽  
Camp Levels

Abstract Using intact human platelets, we studied the effect of sodium fluoride (NaF) on platelet aggregation and release reaction and correlated the functional changes to intracellular events specific for either agonist- induced or antagonist-induced platelet responses. At lower concentrations, with a peak activity between 30 and 40 mmol/L, NaF induced aggregation and release of adenosine 5′-triphosphate (ATP) that was associated with increased formation of inositol phosphates, a rise in cytosolic free Ca2+, and phosphorylation of 20-kd and 40-kd proteins. At NaF concentrations greater than 40 mmol/L, aggregation and ATP release decreased dose-dependently in parallel with a decrease in Ca2+ mobilization, whereas neither inositol phosphate formation nor 40- kd protein phosphorylation was reduced. At these concentrations, NaF caused a dose-dependent transient rise in platelet cyclic adenosine 3′,5′-monophosphate (cAMP) levels that was sufficient to account for the observed reduction in Ca2+ mobilization, aggregation, and ATP release. Stimulated cAMP levels started declining rapidly within 30 seconds of addition of NaF, however. Similarly, prostacyclin (PGI2)- induced cAMP accumulation was temporarily enhanced but subsequently suppressed by NaF, suggesting either stimulation of a cAMP phosphodiesterase or delayed inhibition of adenylate cyclase. Evidence for the latter was provided by the finding that NaF pretreatment of platelets resulted in partial inhibition of PGI2-stimulated cAMP formation in the presence of the cAMP phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (MIX). We conclude that NaF exerts a dual (stimulatory and inhibitory) effect on adenylate cyclase in intact platelets that is accompanied by simultaneous activation of a phosphoinositide-specific phospholipase C; in addition, a cAMP phosphodiesterase may be activated.

scholarly journals The α1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C

1988 ◽  
Vol 253 (1) ◽  
pp. 93-102 ◽  
Author(s):  
R J Schimmel
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Neutral Lipids ◽  
Receptor Activation ◽  
Ionophore A23187 ◽  
Brown Adipocytes ◽  
Diacylglycerol Lipase

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.

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.

Inhibition of TASK-1 potassium channel by phospholipase C

2001 ◽  
Vol 281 (2) ◽  
pp. C700-C708 ◽  
Author(s):  
Gábor Czirják ◽  
Gábor L. Petheő ◽  
András Spät ◽  
Péter Enyedi
Keyword(s):  
Phospholipase C ◽  
Lysophosphatidic Acid ◽  
Protein S ◽  
Receptor Activation ◽  
Xenopus Laevis Oocytes ◽  
Ang Ii ◽  
Inositol 1,4,5 Trisphosphate ◽  
Glomerulosa Cells ◽  
Pore Domain ◽  
Stimulation Of

The two-pore-domain K+ channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca2+-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M1 muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5′- O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca2+ concentration, and diacylglycerol) do not mediate the inhibition. Unlike the Gq-coupled receptors, stimulation of the Gi-activating M2 muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-β2 (which is responsive also to Giβγ-subunits) renders M2 receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

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