scholarly journals Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

2019 ◽  
Vol 119 (07) ◽  
pp. 1124-1137 ◽  
Author(s):  
Joanne C. Clark ◽  
Deirdre M. Kavanagh ◽  
Stephanie Watson ◽  
Jeremy A. Pike ◽  
Robert K. Andrews ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Platelet Activation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Adenosine Receptor Agonist

Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. Objective This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

scholarly journals The effect of pharmacologic inhibition of phospholipid methylation on human platelet function

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 906-912
Author(s):  
SJ Shattil ◽  
JA Montgomery ◽  
PK Chiang
Keyword(s):  
Platelet Activation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Phospholipid ◽  
Biochemical Effect ◽  
Methylation Pathway ◽  
Pharmacologic Inhibition

Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L- methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza- adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza- aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.

scholarly journals The effect of pharmacologic inhibition of phospholipid methylation on human platelet function

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 906-912 ◽  
Author(s):  
SJ Shattil ◽  
JA Montgomery ◽  
PK Chiang
Keyword(s):  
Platelet Activation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Phospholipid ◽  
Biochemical Effect ◽  
Methylation Pathway ◽  
Pharmacologic Inhibition

Abstract Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L- methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza- adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza- aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.

scholarly journals A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels

2000 ◽  
Vol 5 (4) ◽  
pp. 239-247 ◽  
Author(s):  
Anthony C. Chiulli ◽  
Karen Trompeter ◽  
Michelle Palmer
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Wide Range ◽  
G Protein Coupled ◽  
Camp Levels

The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.

scholarly journals β-Arrestin Recruitment Assay for the Identification of Agonists of the Sphingosine 1-Phosphate Receptor EDG1

2008 ◽  
Vol 13 (10) ◽  
pp. 986-998 ◽  
Author(s):  
Miranda M.C. van Der Lee ◽  
Maaike Bras ◽  
Chris J. van Koppen ◽  
Guido J.R. Zaman
Keyword(s):  
Cell Line ◽  
Protein Kinases ◽  
G Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Kinetic Studies ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Cellular Effects ◽  
Extracellular Signal ◽  
Fragment Complementation

β-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter™ assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of β-galactosidase to measure receptor-β-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter™ β-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed β-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter™ cell line at the level of β-arrestin recruitment, Gi/o protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of β-arrestin recruitment was largely unaffected upon blockade of Gi/o protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of β-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully Gi/o protein regulated. The data indicate that the β-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of “biased ligands” (i.e., compounds that differ in G-protein coupling and β-arrestin-mediated cellular effects). ( Journal of Biomolecular Screening 2008:986-998)

scholarly journals Allosteric inhibition of adenylyl cyclase type 5 by G-protein: a molecular dynamics study

2020 ◽  
Author(s):  
Elisa Frezza ◽  
Tina-Méryl Amans ◽  
Juliette Martin
Keyword(s):  
Molecular Dynamics ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Adenosine Triphosphate ◽  
Crucial Role ◽  
Protein A ◽  
Cyclic Adenosine Monophosphate ◽  
Structural Data ◽  
Adenosine Monophosphate ◽  
Protein Subunit

AbstractAdenylyl cyclases (ACs) have a crucial role in many signal transduction pathways, in particular in the intricate control of cyclic AMP (cAMP) generation from adenosine triphosphate (ATP). Using homology models developed from existing structural data and docking experiments, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase bound to the inhibitory G-protein subunit Gαi in the presence and in the absence of ATP. The results show that Gαi have significant effects on the structure and flexibility of adenylyl cyclase, as observed earlier for the binding of ATP and Gsα. New data on Gαi bound to the C1 domain of AC5 help to explain how Gαi inhibits enzyme activity and to get insight on its regulation. Simulations also suggest a crucial role of ATP in the regulation of stimulation and inhibition of AC5.Author summaryThe neurons that compose the human brain are able to respond to multiple inputs from other neurons. The chemical “integration” of these inputs then decides whether a given neuron passes on a signal or not. External chemical messages act on neurons via proteins in their membranes that trigger cascades of reactions within the cell. One key molecule in these signaling cascades is cyclic adenosine monophosphate (cAMP) that is chemically synthesized from adenosine triphosphate (ATP) by the enzyme adenylyl cyclase (AC). We are investigating the mechanisms that control how much cAMP is produced as a function of the signals received by the neuron. In particular, we have studied the inhibition effect of a key protein, termed Gαi, on AC, and we compare it with the stimulator effect of another key protein termed Gsα. Using microsecond molecular simulations, we have been able to show how binding Gαi to AC changes its structure and its dynamics so that its enzymatic activity is quenched and that ATP seems to have a crucial role in the regulation of stimulation and inhibition of AC5.

Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibα mechanoreceptor

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2793-2800 ◽  
Author(s):  
Mario Mazzucato ◽  
Paola Pradella ◽  
Maria Rita Cozzi ◽  
Luigi De Marco ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Activation ◽  
Platelet Adhesion ◽  
Fluid Dynamic ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Activation Process ◽  
Integrin Αiibβ3

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca++ concentration ([Ca++]i) that are associated with sequential stages of integrin αIIbβ3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibα (GPIbα) and the VWF A1 domain led to Ca++ release from intracellular stores (type α/β peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca++]ioscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbα. Once adhesion was established through the integrin αIIbβ3, new [Ca++]i oscillations (type γ) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbα-VWF bonds leads to αIIbβ3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied αIIbβ3, with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbα and αIIbβ3 and lead to platelet adhesion and aggregation on immobilized VWF.

Modulation of the Cardiac Sodium Current by Inhalational Anesthetics in the Absence and Presence of β-Stimulation 

1998 ◽  
Vol 88 (1) ◽  
pp. 114-124 ◽  
Author(s):  
Henry U. Weigt ◽  
Wai-Meng Kwok ◽  
Georg C. Rehmert ◽  
Zeljko J. Bosnjak
Keyword(s):  
G Protein ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Volatile Anesthetic ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Inhibitory Effect ◽  
Specific Protein ◽  
Na Current ◽  
Patch Clamp Technique

Background Cardiac dysrhythmias during inhalational anesthesia in association with catecholamines are well known, and halothane is more "sensitizing" than isoflurane. However, the underlying mechanisms of action of volatile anesthetics with or without catecholamines on cardiac Na channels are poorly understood. In this study, the authors investigated the effects of halothane and isoflurane in the absence and presence of beta-stimulation (isoproterenol) on the cardiac Na+ current (INa) in ventricular myocytes enzymatically isolated from adult guinea pig hearts. Methods A standard whole-cell patch-clamp technique was used. The INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). Results Isoproterenol alone depressed peak INa significantly by 14.6 +/- 1.7% (means +/- SEM). Halothane (1.2 mM) and isoflurane (1.0 mM) also depressed peak INa significantly by 42.1 +/- 3.4% and 21.3 +/- 1.9%, respectively. In the presence of halothane, the effect of isoproterenol (1 microM) was potentiated, further decreasing peak INa by 34.7 +/- 4.1%. The halothane effect was less, although significant, in the presence of a G-protein inhibitor (GDPbetaS) or a specific protein kinase A inhibitor [PKI-(6-22)-amide], reducing peak INa by 24.2 +/- 3.3% and 24 +/- 2.4%, respectively. In combination with isoflurane, the effect of isoproterenol on INa inhibition was less pronounced, but significant, decreasing current by 12.6 +/- 3.9%. GDPbetaS also reduced the inhibitory effect of isoflurane. In contrast, PKI-(6-22)-amide had no effect on isoflurane INa inhibition. Conclusions These results suggest two distinct pathways for volatile anesthetic modulation on the cardiac Na+ current: (1) involvement of G proteins and a cyclic adenosine monophosphate (cAMP)-mediated pathway for halothane and, (2) a G-protein-dependent but cAMP-independent pathway for isoflurane. Furthermore, these studies show that the inhibition of cardiac INa by isoproterenol is enhanced in the presence of halothane, suggesting some form of synergistic interaction between halothane and isoproterenol.

Opposite Effect of Lysine on Platelet Aggregation Induced by Arachidonate and by Other Aggregants

1982 ◽  
Vol 48 (01) ◽  
pp. 078-083 ◽  
Author(s):  
C Ts'ao ◽  
S J Hart ◽  
D V Krajewski ◽  
P G Sorensen
Keyword(s):  
Platelet Aggregation ◽  
Secondary Phase ◽  
Aminocaproic Acid ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Primary Phase ◽  
High Doses ◽  
The Many ◽  
Induced Aggregation

SummaryEarlier, we found that ε-aminocaproic acid (EACA) inhibited human platelet aggregation induced by adenosine diphosphate (ADP) and collagen, but not aggregation by arachidonic acid (AA). Since EACA is structurally similar to lysine, yet these two agents exhibit vast difference in their antifibrinolytic activities, we chose to study the effect of lysine on platelet aggregation. We used L-lysine-HCl in these studies because of its high solubility in aqueous solutions while causing no change in pH when added to human plasma. With lysine, we repeatedly found inhibition of ADP-, collagen- and ristocetin-induced aggregation, but potentiation of AA-induced aggregation. Both the inhibitory and potentiation effects were dose-dependent. Low doses of lysine inhibited the secondary phase of aggregation; high doses of it also inhibited the primary phase of aggregation. Potentiation of AA-induced aggregation was accompanied by increased release of serotonin and formation of malondialdehyde. These effects were not confined to human platelets; rat platelets were similarly affected. Platelets, exposed to lysine and then washed and resuspended in an artificial medium not containing lysine, remained hypersensitive to AA, but no longer showed decreased aggregation by collagen. Comparing the effects of lysine with equimolar concentrations of sucrose, EACA, and α-amino-n-butyric acid, we attribute the potent inhibitory effect of lysine to either the excess positive charge or H+ and C1− ions. The -NH2 group on the α-carbon on lysine appears to be the determining factor for the potentiation effect; the effect seems to be exerted on the cyclooxygenase level of AA metabolism. Lysine and other chemicals with platelet-affecting properties similar to lysine may be used as a tool for the study of the many aspects of a platelet aggregation reaction.

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

2019 ◽  
Author(s):  
Bella Grigorenko ◽  
Igor Polyakov ◽  
Alexander Nemukhin
Keyword(s):  
Adenylyl Cyclase ◽  
Bond Cleavage ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Md Simulations ◽  
Coordination Shell ◽  
Energy Profile ◽  
One Step ◽  
Type V

<p>We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of Mg<sub>A</sub><sup>2+ </sup>are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of Mg<sub>A</sub><sup>2+ </sup>does not include the O<sub>δ1</sub> atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O<sup>3'</sup>H<sup>3' </sup>group in ATP to Asp440 via a shuttling water molecule and P<sup>A</sup>-O<sup>3A</sup> bond cleavage and O<sup>3'</sup>-P<sup>A</sup> bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, Mg<sub>A</sub><sup>2+</sup> is bound to the O<sub>δ1</sub>(Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O<sup>3'</sup>H<sup>3' </sup>group via shuttling water molecules. </p>

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