Protein kinase C ε-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells

Chen, Bing Chang; Lin, Lih Ling; Lin, Wan-Wan

doi:10.1016/s0014-2999(99)00238-1

Cited by 17 publications

(22 citation statements)

References 33 publications

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“…A number of studies have examined desensitization of P2Y 1 purinergic receptors in a variety of settings, including neuronal and endothelial cells. 39,40 In agreement with our investigation, they also concluded that this receptor undergoes rapid desensitization, consistent with regulation by kinases.Two families of protein kinases are predominantly involved in the regulation of GPCRs: GRKs, which phosphorylate agonistoccupied GPCRs to mediate homologous receptor desensitization, 41 and second messenger-activated kinases, such as PKC, which phosphorylate ligand-bound and inactive GPCRs in a heterologous manner. 42,43 In this study we decided to overexpress catalytically dead dominant-negative GRK constructs to block endogenous GRK activity 41 and to reduce endogenous GRK expression by the use of specific siRNAs.…”

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confidence: 90%

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P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

Hardy

Conley

Luo

et al. 2005

Blood

159

205

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Adenosine 5-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y 1 and P2Y 12 . Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y 1 -and P2Y 12 -mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y 12 , but not P2Y 1 , desensitization is mediated by GRKs. In contrast, desensitization of P2Y 1 , but not P2Y 12 , is largely dependent on protein kinase C activity. This study is the first to show that both P2Y 1 and P2Y 12 desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered. IntroductionPlatelets are activated by a variety of extracellular stimuli and are an essential component of the normal response to vascular injury. Central among these stimuli is adenosine 5Ј-diphosphate (ADP), which induces multiple platelet responses and potentiates platelet aggregation to other agonists (for reviews, see Gachet 1 and Kunapuli et al 2 ). Indeed, since it was recognized 40 years ago, ADP has been regarded as a central mediator of hemostasis and thrombosis by providing a positive feedback mechanism for the activation of platelets by multiple agonists. For example, both thrombin and collagen promote ADP release from platelet-dense granules; ADP subsequently acts on purinergic receptors to reinforce platelet aggregation responses 1,2 and thrombus formation.ADP acts on 2 G protein-coupled receptors (GPCRs), P2Y 1 and P2Y 12 . 1,2 The P2Y 1 purinergic receptor was the first of the ADP receptors to be cloned. [3][4][5] This receptor is widely expressed throughout the body and couples to G q , leading to the activation of phospholipase C␤, a subsequent increase in cytosolic calcium, and the activation of protein kinase C (PKC). The P2Y 12 receptor was only recently identified 6 and is the target of the clinically effective antithrombotic drugs clopidogrel and ticlopidine. P2Y 12 couples through G i to the inhibition of adenylyl cyclase and the activation of PI3-kinase. On the basis of pharmacologic and genetic studies, it is now accepted that P2Y 1 is required for platelet activation by ADP, whereas P2Y 12 is important in synergizing with P2Y 1 or other G q -coupled receptors to induce platelet activation by ADP and other agonists playing a major role in stabilizing platelet thrombi in vivo.Given the established crucial role of ADP in platelet activation, it is likely that the responsiveness of platelets to ADP is tightly regulated, and knowledge of the mechanisms responsible for t...

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confidence: 90%

supporting

confidence: 90%

P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

Hardy

Conley

Luo

et al. 2005

Blood

159

205

View full text Add to dashboard Cite

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“…The results that PKC activator PMA-elicited complete phosphorylation of ERK and cPLA 2 are additive to thapsigargin responses indicate the presence of the latter assumption, i.e., thapsigargin-induced cPLA 2 phosphorylation has already played its permissive role in enzyme activity, and thereby PMA cannot further enhance thapsigargin-induced AA release. This phenomenon is unique from those observed in different cell types, such as in endothelial cells [Chen et al, 1999] and macrophages [Lin and Chen, 1998a,b;Ambs et al, 1995] where full cPLA 2 activity is achieved by co-stimulation with Ca 2ϩ -elevating agent and PMA. Apparently one reason for the potentiation effect is ascribed to the inability of Ca 2ϩ -elevating agent itself to induce ERK and cPLA 2 phosphorylation in these cells [Chen et al, 1999;Ambs et al, 1995].…”

Section: Discussionmentioning

confidence: 75%

“…As described previously [Chen et al, 1999], cells in 24-well plates (approximately 3 ϫ 10 5 cells/well) were labeled with 0.3 Ci/ml [ 3 H]AA in DMEM overnight. Cells were then washed three times with serum-free DMEM and incubated in medium containing 0.5% fatty acidfree bovine serum albumin for 20 min before stimulation with thapsigargin for different intervals as indicated.…”

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confidence: 99%

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Basal cPLA2 phosphorylation is sufficient for Ca2+-induced full activation of cPLA2 in A549 epithelial cells

Wen

Lin

2000

J. Cell. Biochem.

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The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.

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Regulation of vascular endothelial cells and vascular smooth muscle cells by multiple P2Y receptor subtypes

Boarder

White

Roberts

et al. 2001

Drug Development Research

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Receptors for nucleotides regulate all aspects of vascular function. Here, the role and function of P2Y receptors in vascular endothelium and smooth muscle cell proliferation are explored. In both cell types the presence of multiple P2Y receptors is commonplace; however, the identity and role of these receptors is dependent on the vessel of origin. This is clearly seen with endothelial cells. The commonest observation is coexisting P2Y 1 and P2Y 2 (or P2Y 4 ) receptors coupled through inositol 1,4,5-trisphosphate production to Ca 2+ mobilisation. However, in some endothelial cells other P2Y receptors are present. Brain microvascular endothelial cells, for example, exhibit a variety of responses with profiles which cannot be fully explained by cloned P2Y receptors, and which are coupled to diverse receptor-effector mechanisms. These include p42/p44 mitogen-activated protein kinase activation, enhanced cyclic AMP synthesis and raised cytosolic Ca 2+ in the absence of detectable rises in inositol 1,4,5-trisphosphate. These responses can be expected to influence endothelial prostacyclin and nitric oxide production, permeability, adhesion factor expression, and leukocyte-endothelial interaction. P2Y receptors also regulate proliferative responses. Previously, data has been presented indicating that nucleotides acting on P2Y receptors can enhance cell proliferation in response to other agents. Here, we discuss evidence that UTP is an antiproliferative regulator in human vascular smooth muscle cells and that P2Y 4 receptors may be involved in this response. Drug Dev.

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Protein kinase C ε-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells

Cited by 17 publications

References 33 publications

P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

Basal cPLA2 phosphorylation is sufficient for Ca2+-induced full activation of cPLA2 in A549 epithelial cells

Regulation of vascular endothelial cells and vascular smooth muscle cells by multiple P2Y receptor subtypes

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