Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.     

Toll-like receptor 4-mediated cAMP production up-regulates B-cell activating factor expression in Raw264.7 macrophages

B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression.     

ROS/Epac1-mediated Rap1/NF-kappaB activation is required for the expression of BAFF in Raw264.7 murine macrophages

B-cell activating factor (BAFF) plays a role for the maturation and the maintenance of B cells. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction, which resulted in BAFF expression through nuclear factor kappa B (NF-κB) activation. Here, we investigated whether BAFF expression could be regulated by p65 phosphorylation through the production of reactive oxygen species (ROS) or cyclic AMP (cAMP) in Raw264.7 murine macrophages. mBAFF expression was reduced by ROS scavengers and it was increased by dibutyl-cAMP, a cAMP analogue. mBAFF expression and mBAFF promoter activity were increased by co-transfection of p65 but they were reduced by p65-small interference (si) RNA. Serine (Ser) 276 phosphorylation of p65 was increased by LPS-mediated PKA activation or by the treatment with forskolin, adenylate cyclase activator and dibutyl-cAMP. In contrast, p65 phosphorylation at Ser276 was decreased by ROS scavengers. H₂O₂ increased intracellular cAMP concentration, significantly. While no increase in p65 phosphorylation at Ser276 was detected by the treatment with H₂O₂, CREB and p65 phosphorylation at Ser133 and Ser536 was observed, respectively. It implicates that p65 phosphorylation at Ser276 is independent of ROS-induced cAMP production. As another cAMP effector protein was cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor, NF-κB was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT) that is an activator to Epac. Epac1-mediated Rap1 was activated by the treatment with H₂O₂ but it was inhibited by ROS scavengers. CPT induced p65 phosphorylation at both Ser276 and Ser536. CPT also increased not only mBAFF expression but mBAFF promoter activity. Data demonstrate that TLR4-mediated mBAFF expression was resulted from the crosstalk of p65 phosphorylation at Ser536 and Ser276 through ROS- and/or cAMP-mediated signal transduction. It suggests for the first time that ROS/Epac1-mediated Rap1/NF-κB pathway could be required for BAFF expression.     

Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications

Epac proteins respond to the second messenger cyclic AMP (cAMP) and are activated by Gs coupled receptors. They act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. A plethora of studies using 8-pCPT-2′-O-Me-cAMP, an Epac agonist, has revealed the importance of these multi-domain proteins in the control of key cellular functions such as cell division, migration, growth and secretion. Epac and protein kinase A (PKA) may act independently but are often associated with the same biological process, in which they fulfill either synergistic or opposite effects. In addition, compelling evidence is now accumulating about the formation of molecular complexes in distinct cellular compartments that influence Epac signaling and cellular function. Epac is spatially and temporally regulated by scaffold protein and its effectors are interconnected with other signaling pathways. Pathophysiological changes in Epac signaling may underlie certain diseases.     

cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Identification and Validation of Modulators of Exchange Protein Activated by cAMP (Epac) Activity: STRUCTURE-FUNCTION IMPLICATIONS FOR Epac ACTIVATION AND INHIBITION*

The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2′-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (R_p)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.     

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl⁻ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca²⁺]_i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl⁻ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I_sc) measurement in response to agonist-stimulated Cl⁻ secretion. FSK-stimulated Cl⁻ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca²⁺]_i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca²⁺]_i, activated Ras-related protein 2, and induced Cl⁻ secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I_sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl⁻ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl⁻ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl⁻>Br⁻>I⁻ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca²⁺]_i signaling pathway is involved in cAMP-stimulated Cl⁻ secretion, which is carried by a novel, previously undescribed Cl⁻ channel.     

PDE7A1 hydrolyzes cCMP

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP K_M value of 135 ± 19 μM. The V_max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.     

Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E(2) -induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c-Myc and VEGF expression

Prostaglandin E₂ (PGE₂) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE₂‐induced hUCB‐MSC proliferation and its related signaling pathways. PGE₂ increased cell proliferation, and E‐type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE₂ increased Epac1 expression, Ras‐related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1‐specific siRNA. Also, PGE₂ increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB‐MSCs were incubated with the Epac agonist 8‐pCPT‐cAMP or the PKA agonist 6‐phe‐cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8‐pCPT‐cAMP increased Akt phosphorylation but not PKA activity. 6‐Phe‐cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE₂‐induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE₂ increased glycogen synthase kinase (GSK)‐3β phosphorylation and nuclear translocation of active‐β‐catenin, which were inhibited by Akt inhibitor or/and PKI. PGE₂ increased c‐Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β‐catenin siRNA. In conclusion, PGE₂ stimulated hUCB‐MSC proliferation through β‐catenin‐mediated c‐Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation. J. Cell. Physiol. 227: 3756–3767, 2012. © 2012 Wiley Periodicals, Inc.     

Cytotoxic T lymphocyte antigen-2 alpha induces apoptosis of murine T-lymphoma cells and cardiac fibroblasts and is regulated by cAMP/PKA

The mechanism of cAMP-promoted apoptosis is not well defined. In wild-type (WT) murine S49 lymphoma cells, cAMP promotes apoptosis in a protein kinase A (PKA)-dependent manner. We find that treatment of WT S49 cells with 8-CPT-cAMP prominently increases the expression (as determined by DNA microarray analysis, real-time PCR and immunblotting) of cytotoxic T lymphocyte antigen-2α (CTLA-2α), a cathepsin L-like cysteine protease inhibitor. By contrast, CTLA-2α expression is only slightly increased by 8-CPT-cAMP treatment of D-S49 cells, which lack cAMP/PKA-promoted apoptosis. Raising endogenous cAMP (by use of forskolin or inhibition of phosphodiesterase [PDE] 4) or a PKA-selective, but not an Epac-selective, cAMP analogue, increases CTLA-2α mRNA expression; PKA, and not Epac, thus mediates the increase in CTLA-2α expression. An adenoviral CLTA-2α (Ad-CTLA-2α) construct induces apoptosis and enhances cAMP-promoted apoptosis in WT S49 cells but such cells do not have an increase in cathepsin L activity nor does a cathepsin L inhibitor alter cAMP-promoted apoptosis. 8-CPT-cAMP also increases CTLA-2α expression and induces apoptosis in murine cardiac fibroblasts; knockdown of CTLA-2α expression by siRNA blocks 8-CPT-cAMP-promoted apoptosis. Thus, cAMP increases CTLA-2α expression in murine lymphoma and cardiac fibroblasts and this increase in CTLA-2α contributes to cAMP/PKA-promoted apoptosis by mechanisms that are independent of the ability of CTLA-2α to inhibit cathepsin L.     

5,7,3′,4′-Tetramethoxyflavone exhibits chondroprotective activity by targeting β-catenin signaling in vivo and in vitro

Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. This study is the first report which demonstrates the cartilage protective effect of 5,7,3′,4′-tetramethoxyflavone (TMF) by decreasing the concentration of IL-1β, TNF-α and PGE₂ in the knee synovial fluid in OA rat models in vivo. In vitro, after induced by PGE₂, the apoptosis rate of chondrocytes was significantly increased. In addition, PGE₂ increased the expression of cAMP/PKA signaling pathway in chondrocytes, stabilized and accumulated β-catenin, and activated the expression of β-catenin signaling pathway. These activities were counteracted by TMF dose-dependently. Collectively, TMF is a potential compound with chondroprotective activity by inhibiting both EP/cAMP/PKA signaling pathway and β-catenin signaling pathway.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

β-Adrenergic Receptors Activate Exchange Protein Directly Activated by cAMP (Epac), Translocate Munc13-1, and Enhance the Rab3A-RIM1α Interaction to Potentiate Glutamate Release at Cerebrocortical Nerve Terminals

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na⁺ channels with tetrodotoxin. We found that 8-pCPT-2′-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

The P2Y12 Antagonists, 2MeSAMP and Cangrelor,Inhibit Platelet Activation through P2Y12/Gi-Dependent Mechanism

Background

ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y₁ (Gα_q-coupled) and P2Y₁₂ (Gα_i-coupled). P2Y₁₂ plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y₁₂ antagonists, 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y₁₂ in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y₁₂-independent mechanism.

Methodology/Principal Findings

The present work, using P2Y₁₂ deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y₁₂ deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI₂ and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca²⁺ mobilization, Akt phosphorylation, and Rap1b activation in P2Y₁₂ deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl₃-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y₁₂ deficient mice.

Conclusions

These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y₁₂-dependent mechanism both in vitro and in vivo.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

The Epac1 Signaling Pathway Regulates Cl? Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

The apical membrane of intestinal epithelia expresses intermediate conductance K⁺ channel (KCNN4), which provides the driving force for Cl⁻ secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl⁻ secretion via stimulation of Rap2-phospholipase Cϵ-[Ca²⁺]_i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl⁻ secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl⁻ secretion and apical K⁺ conductance (I_K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′-O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K⁺ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I_K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I_K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.