[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid (BMY 45778) is a potent non-prostanoid prostacyclin partial agonist: Effects on platelet aggregation, adenylyl cyclase, cAMP levels, protein kinase, and iloprost binding

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca2+release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.

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Regulation of Protein Kinase C by the Platelet P2Y12 ADP Receptor.

Blood ◽

10.1182/blood.v106.11.1647.1647 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1647-1647

Author(s):

Paolo Lova ◽

Gianni F. Guidetti ◽

Francesca Campus ◽

Bruno Bernardi ◽

Cesare Balduini ◽

...

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Adenylyl Cyclase ◽

Thromboxane A2 ◽

Diacylglycerol Kinase ◽

P2y12 Receptor ◽

Kinase C ◽

Stimulation Of

Abstract The Gi-coupled P2Y12 receptor plays a crucial role in platelet activation induced by ADP, and is also required for the action of a number of agonists whose receptors are couple to Gq, such as thromboxane A2. Stimulation of P2Y12 leads to the inhibition of adenylyl cyclase through the Gai subunit, and to the activation of phosphatidylinositol 3-kinase (PI-3K) and Rap1b through the bg subunit. Pharmacological inhibition of P2Y12 receptor results in the prevention of platelet aggregation induced by a number of agonists, including ADP and the the thromboxane A2 analogue U46619. In this study, we report that P2Y12 receptor is absolutely necessary for the stimulation of protein kinase C (PKC) and for PKC-directed protein phosphorylation. By using 32P-labeled human platelets, as well as by immunoblotting with anti-phospho(Ser) PKC substrates antibody, we analyzed the phosphorylation of pleckstrin, the main platelet substrate for PKC. As expected, we found that ADP-induced pleckstrin phosphorylation was totally suppressed by antagonists of the Gq-coupled P2Y1 receptor, but, surprisingly, we found that it was also completely prevented upon blockade of the P2Y12 receptor by AR-C69931MX or by 2-MeSAMP. Although we failed to directly detect any production of IP3 in ADP-stimulated platelets, we observed that ADP-induced Ca2+ release from intracellular stores was not affected by P2Y12 receptor antagonists. Moreover, AR-C69931MX or 2-MeSAMP strongly impaired pleckstrin phosphorylation induced by U46619, without affecting intracellular Ca2+ mobilization. These results indicated that blockade of the P2Y12 receptor did not interfere with phospholipase C stimulation, and that the reduced activation of PKC is probably not the consequence of a reduced production of diacylglycerol. Experiments performed with specific cell permeable inhibitors demonstrated that, in ADP-treated platelets, neither inhibition of adenylyl cyclase nor activation of PI-3K downstream P2Y12 receptor stimulation was responsible for PKC regulation. Subthreshold concentrations of PMA were able to restore ADP- or U46619-induced pleckstrin phosphorylation in the presence of P2Y12 antagonists, but were unable to restore platelet aggregation. Moreover, direct inhibition of PKC by Ro31-8220 prevented ADP-induced pleckstrin phosphorylation, but aggregation occurred normally. These results indicate that although PKC-directed protein phosphorylation is targeted by the P2Y12 receptor, it is not responsible for the essential contribution of this receptor to platelet aggregation. By contrast, we found that inhibition of platelet diacylglycerol kinase by R59949 completely reverted the inhibitory effect of AR-C69931MX or 2-MeSAMP on ADP- and U46619-induced activation of PKC, and completely restored platelet aggregation. These results suggest that diacylglycerol kinase may be regulated downstream the Gi-coupled P2Y12 receptor, and may be an important new regulator of platelet aggregation.

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Role of Intracellular Signaling Events in ADP-induced Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614384 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1322-1326 ◽

Cited By ~ 84

Author(s):

James Daniel ◽

Carol Dangelmaier ◽

Jianguo Jin ◽

Young Kim ◽

Satya Kunapuli

Keyword(s):

Platelet Aggregation ◽

Adenylyl Cyclase ◽

Phospholipase C ◽

Intracellular Signaling ◽

Receptor Activation ◽

Human Platelets ◽

Signaling Events ◽

Adp Receptors ◽

G Protein Coupled ◽

Camp Levels

SummaryHuman platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the α2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5’-phosphate-3’-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to αIIbβ3 activation.

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Protein kinases A and C regulate receptor-mediated increases in cAMP in rabbit erythrocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00975.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. H587-H593 ◽

Cited By ~ 10

Author(s):

Shaquria P. Adderley ◽

Meera Sridharan ◽

Elizabeth A. Bowles ◽

Alan H. Stephenson ◽

Mary L. Ellsworth ◽

...

Keyword(s):

Protein Kinase ◽

Adenylyl Cyclase ◽

Protein Kinases ◽

Adrenergic Receptor ◽

Atp Release ◽

Pde4 Inhibitor ◽

Calphostin C ◽

Prostacyclin Receptor ◽

Camp Levels ◽

Pde3 Inhibitor

Activation of the β-adrenergic receptor (β-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs ( 1 ). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μM), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway.

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Ticlopidine and Clopidogrel (SR 25990C) Selectively Neutralize ADP Inhibition of PGE1-Activated Platelet Adenylate Cyclase in Rats and Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647481 ◽

1991 ◽

Vol 65 (02) ◽

pp. 186-190 ◽

Cited By ~ 61

Author(s):

G Defreyn ◽

C Gachet ◽

P Savi ◽

F Driot ◽

J P Cazenave ◽

...

Keyword(s):

Platelet Aggregation ◽

Adenylate Cyclase ◽

Direct Effect ◽

Cyclic Nucleotide ◽

Camp Accumulation ◽

Rabbit Platelets ◽

Camp Levels ◽

Kinetics Of ◽

Camp Elevation ◽

Rat Platelets

SummaryTiclopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGEj-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGEr stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGEX-induced cAMP elevation but this effect seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGEr activated platelet adenylate cyclase in rats and rabbits.

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Synergistic Antiplatelet Effect of Ridogrel, a Combined Thromboxane Receptor Antagonist and Thromboxane Synthase Inhibitor, and UDCG-212, a cAMP-Phosphodiesterase Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649677 ◽

1993 ◽

Vol 70 (05) ◽

pp. 822-825 ◽

Cited By ~ 7

Author(s):

B Hoet ◽

J Arnout ◽

H Deckmyn ◽

J Vermylen

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Receptor Antagonist ◽

Phosphodiesterase Inhibitor ◽

Camp Phosphodiesterase ◽

Thromboxane Receptor ◽

Thromboxane Synthase Inhibitor ◽

Thromboxane Receptor Antagonist ◽

Synthase Inhibitor ◽

Camp Levels

SummaryRidogrel, a combined thromboxane receptor antagonist and thromboxane synthase inhibitor (1), inhibits platelet aggregation. Following stimulation with arachidonic acid, cAMP-levels are increased in human platelets preincubated with ridogrel, this is due to the known reorientation of the metabolism of the formed endoperoxides towards adenylate cyclase stimulating prostaglandins.Pretreatment of resting platelets with UDCG-212, a cAMP-phosphodiesterase inhibitor (2), also inhibits platelet aggregation induced by arachidonic acid, concomitant with an increase in cAMP levels, due to an inhibition of its breakdown. Under basal conditions, cAMP also is increased.By combining the two drugs, a more than additive action was observed on platelet aggregation and on both resting and stimulated platelet cAMP content. The appropriate combination may result in a more effective antiplatelet strategy.

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Effects of parathyroid hormone and agonists of the adenylyl cyclase and protein kinase C pathways on bone cell proliferation

Bone ◽

10.1016/8756-3282(95)00427-0 ◽

1996 ◽

Vol 18 (1) ◽

pp. 59-65 ◽

Cited By ~ 30

Author(s):

M. Sabatini ◽

C. Lesur ◽

M. Pacherie ◽

P. Pastoureau ◽

N. Kucharczyk ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Kinase C ◽

Parathyroid Hormone ◽

Protein Kinase ◽

Adenylyl Cyclase ◽

Bone Cell ◽

Kinase C ◽

Bone Cell Proliferation

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Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases

Biochemical Journal ◽

10.1042/bj2580057 ◽

1989 ◽

Vol 258 (1) ◽

pp. 57-65 ◽

Cited By ~ 32

Author(s):

W Siess ◽

E G Lapetina

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Protein Kinases ◽

Shape Change ◽

Ionophore A23187 ◽

Maximal Effect ◽

Kinase C ◽

Dependent Protein ◽

High Concentrations

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

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