Short communicationExtraction of cyclic amp for the determination in the competitive protein binding assay
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Cited by (22)
P2Y<inf>12</inf> a new platelet ADP receptor, target of clopidogrel
2001, Biochemical and Biophysical Research CommunicationsThe binding characteristics of 33P-2MeS-ADP, a stable analogue of ADP, were determined on CHO cells transfected with the human P2Y12 receptor, a novel purinergic receptor. These transfected CHO cells displayed a strong affinity for 33P-2MeS-ADP, the binding characteristics of which corresponded in all points to those observed on platelets. In particular, this receptor recognised purines with the following order of potency: 2MeS-ADP = 2MeS-ATP > ADP = ATPγS = ATP ⪢ UTP, a binding profile which is similar to that obtained in platelets. The binding of 33P-2MeS-ADP was antagonised by pCMPS but not by MRS2179 and FSBA, antagonists of P2Y1 and aggregin, respectively. Moreover, the binding of 33P-2MeS-ADP to these cells was strongly and irreversibly inhibited by the active metabolite of clopidogrel with a potency which was consistent with that observed for this compound on platelets. Like in platelets, 2MeS-ADP induced adenylyl cyclase down-regulation in these P2Y12 transfected CHO cells, an effect which was absent in the corresponding non-transfected cells. As already shown in platelets, the active metabolite of clopidogrel antagonised 2MeS-ADP-induced inhibition of adenylyl cyclase on transfected cells. Our results confirm that P2Y12 is the previously called “platelet P2tAC” receptor and show that this receptor is antagonised by the active metabolite of clopidogrel.
Role of P2Y1 purinoceptor in ADP-induced platelet activation
1998, FEBS LettersADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.
Defect in potentiation of adenylyl cyclase correlates with bronchial hyperreactivity
1995, The Journal of Allergy and Clinical ImmunologyBackground: Adenylyl cyclase is a transmembrane signaling system involved in the inhibition of cellular responses. Recently, we showed that the activity of adenylyl cyclase may be potentiated by stimuli that induce an increase of cellular responses but that do not activate adenylyl cyclase. This is probably an important physiologic feedback mechanism that prevents cells from becoming “overstimulated.” Objective: Because increased cellular activities are frequently observed in persons with asthma, we hypothesized that a defect in potentiation of adenylyl cyclase might be involved. Methods: Potentiation of isoprenaline-induced adenosine cyclic monophosphate (cAMP) production with the mitogen phytohemagglutinin (PHA; 45 μg/ml) or the calcium ionophore A23187 (1 μmol/L) was studied in peripheral blood mononuclear cells taken from patients with asthma (n = 8) and healthy control subjects (n = 11). Results: Isoprenaline-induced cAMP production was potentiated significantly in the healthy control subjects (PHA, 110% ± 15%; A23187, 92% ± 25%). In contrast, potentiation was not seen with PHA or A23187 in the total group of patients with asthma. However, some patients showed weak potentiation, whereas in others PHA decreased isoprenaline-induced cAMP production. Moreover, the effect of PHA on isoprenaline-induced cAMP production correlated significantly with the degree of bronchial hyperreactivity in patients with asthma (r = 0.96; p = 0.0001). Conclusions: The observed defect in signal transduction could play an important part in bronchial hyperresponsiveness. (J ALLERGY CLIN IMMUNOL 1995;96:628-34.)
Evidence for the existence of two different ADP-binding sites on rat platelets
1994, Thrombosis Research[3H]-2-Methylthio-ADP ( [3H]-2-MeS-ADP ), a stable analogue of ADP bound to one type of specific binding sites on rat platelets ( KD = 0.77 ± 0.07 nM, Bmax = 160 ± 11 fmol/108 cells ). 2-MeS-ADP and ADP antagonized [3H]-2-MeS-ADP binding, showing respective Ki values of 1.4 ± 0.1 nM and 486 ± 78 nM. Clopidogrel, a potent and specific inhibitor of ADP-induced platelet aggregation partially inhibited ( ∼70% inhibition ) the binding of [3H]-2-MeS-ADP at the same time it abrogated 2-MeS-ADP-and ADP-induced adenylyl cyclase inhibition and aggregation. A population of clopidogrel-resistant [3H]-2-MeS-ADP binding sites was detected on platelets from treated animals. These receptor sites ( KD = 0.9 ± 0.2 nM, Bmax = 47 ± 5 fmol/108 platelets ) which showed high affinity for both ADP and 2-MeS-ADP (Ki values in the nanomolar range) might be involved in the ADP-induced shape change, a clopidogrel-resistant ADP-induced event. Using clopidogrel which acts via a direct and irreversible inhibition of ADP binding to its adenylyl cyclase-coupled receptor sites on platelets, we were able to discriminate between two types of ADP receptor sites.
The former which was clopidogrel-sensitive represented about 70% of the total [3H]-2-MeS-ADP receptors and was responsible for ADP-induced platelet aggregation and adenylyl cyclase inhibition. The latter which was not affected by clopidogrel might be involved in ADP-induced shape-change.
Potentiation of adenylyl cyclase in human peripheral blood mononuclear cells by cell-activating stimuli
1994, Biochemical PharmacologyThe isoprenaline-induced production of cAMP in human peripheral blood mononuclear cells (PBMC) was potentiated significantly by incubating PBMC with isoprenaline in the presence of phytohaemagglutinin (PHA), Concanavalin A (Con A) or A23187. This potentiation, that proved to be dependent on the concentration of PHA, Con A or A23187, increased the maximal response but did not cause a change in the potency of isoprenaline. Potentiation could not be induced by the phorbol ester phorbolmyristate acetate, suggesting that protein kinase C-dependent pathways are not likely to be involved in potentiation of adenylyl cyclase. Potentiation could be inhibited by chelating extracellular Ca2+ with EGTA and also by N-(6-aminohexyl)-5-chloro-l-naphtalenesulfonamine, an inhibitor of calmodulin. Potentiation could not be induced by preincubation of PBMC with PHA, suggesting that transient biochemical changes are involved. It was concluded from these results that potentiation in PBMC probably involves the activation of Ca2+/calmodulin-dependent adenylyl cyclase subtypes. Potentiation of the adenylyl cyclase activity could be an important physiological mechanism in vivo preventing cells from becoming “over stimulated”.
Desensitization of the adenylyl cyclase system in peripheral blood mononuclear cells from patients with asthma three hours after allergen challenge
1993, The Journal of Allergy and Clinical ImmunologyBackground: Bronchial hyperreactivity is a common characteristic of patients with asthma and is often associated with enhanced activities of peripheral blood cells. Signal transduction systems are important in regulating cellular activities and can be modified by allergen challenge.
Methods: Patients with allergic asthma (n = 15) were challenged with house dust mite allergen, resulting in an asthmatic response. Adenylyl cyclase activity was measured in membranes from peripheral blood mononuclear cells before, 3 hours after, and 24 hours after challenge.
Results: Allergen challenge proved to have opposite effects in two distinct subgroups of patients. In 10 patients (group I) a heterologous desensitization of the adenylyl cyclase system was observed after challenge, whereas in five patients (group II) an increase in adenylyl cyclase activity was found. Adenylyl cyclase activity before allergen challenge in group II was significantly lower than in group I and comparable to cyclase activity found in group I after allergen challenge. This suggests that in these five patients the adenylyl cyclase system was already desensitized before the start of the study, possibly as a result of natural allergen exposure. Heterologous desensitization in group I was found within 3 hours after allergen challenge, that is before the onset of the late bronchoconstrictive reaction.
Conclusions: Because adenylyl cyclase is important in the regulation of cytokine production by mononuclear cells, alteration of cytokine production induced by desensitization of adenylyl cyclase could therefore play a role in the development of the late bronchoconstrictive reaction.