Modulation of A2a receptor antagonist on D2 receptor internalization and ERK phosphorylation

Aim:

To explore the effects of heterodimerization of D₂ receptor/A_2a receptor (D₂R/A_2aR) on D₂R internalization and D₂R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A_2aR and D₂R in vitro.

Methods:

Primary cultured rat striatal neurons and HEK293 cells co-expressing A_2aR and D₂R were treated with A_2aR- or D₂R-specific agonists. D₂R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A_2aR and D₂R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation.

Results:

D₂R and A_2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D₂R agonist quinpirole (1 μmol/L) marked increased Src phosphorylation and β-arrestin recruitment, thereby D₂R internalization. Co-treatment with the A_2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D₂R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 μmol/L) blocked D₂R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin (319-418) significantly attenuated D₂R-mediated ERK phosphorylation in HEK293 cells expressing both D₂R and A_2aR, but not in those expressing D₂R alone.

Conclusion:

A_2aR antagonist ZM241385 significantly attenuates D₂R internalization and D₂R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A_2aR/D₂R heterodimerization plays important roles in D₂R downstream signaling.     

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin

BACKGROUND AND PURPOSE

A_2B adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.

EXPERIMENTAL APPROACH

HEK 293 cells stably transfected with human A_2B adenosine receptors (HEK-A_2B) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.

KEY RESULTS

In HEK-A_2B cells, the selective A_2B receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G_i or G_o proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.

CONCLUSION AND IMPLICATIONS

Activation of A_2B adenosine receptors reduced superoxide generation from mitochondrial complex I through G_i/o, ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.     

Mitogen-activated protein kinase (MAPK) pathway mediates the oestrogen-like activities of ginsenoside Rg1 in human breast cancer (MCF-7) cells

Background and purpose:

The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen-like actions in MCF-7 cells by activating the mitogen-activated protein kinase (MAPK) pathway in a ligand-independent manner.

Experimental approach:

MCF-7 cells were co-incubated with the MAPK inhibitor PD98059 to determine whether the stimulant effects of Rg1 on cell proliferation, the induction of IGF-IR and pS2, the functional transactivation of oestrogen receptor-α (ERα), as well as ERα phosphorylation are dependent on MAPK. The time-dependent responses of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) to Rg1 in MCF-7 cells were studied. The responses of MEK phosphorylation to Rg1 in oestrogen receptor (ER)-negative HEK293 cells were also determined. The effects of Rg1 on cell proliferation and IGF-IR protein expression were studied in the presence of tyrosine kinase inhibitor genistein to elucidate the involvement of tyrosine kinase in mediating these effects.

Key results:

The oestrogenic effects of Rg1 in MCF-7 cells were abolished in the presence of PD98059. Rg1 could induce MEK protein expression and the phosphorylation level of MEK and ERK significantly in a time- and dose-dependent manner. Rg1 activated MEK phosphorylation in ER-negative HEK293 cells in a time- and dose-dependent manner. Rg1 induction of cell proliferation and IGF-IR protein expression was abolished by co-treatment with genistein.

Conclusions and implications:

Taken together, these results show that the MAPK pathway is involved in mediating the oestrogen-like actions of Rg1 in MCF-7 cells and suggest that Rg1 may activate ERα via MEK/ERK in a ligand-independent manner.     

JWH018, a common constituent of ��Spice�� herbal blends, is a potent and efficacious cannabinoid CB1 receptor agonist

Background and purpose:

‘Spice’ is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis-like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of ‘Spice’. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB₁) cannabinoid receptor and inhibits cAMP production in CB₁ receptor-expressing CHO cells. Other effects of JWH018 on CB₁ receptor-mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB₁ receptors.

Experimental approach:

We investigated the effects of JWH018 on neurotransmission in cultured autaptic hippocampal neurons. We further analysed its activation of ERK1/2 mitogen activated protein kinase (MAPK) and internalization of CB₁ receptors in HEK293 cells stably expressing this receptor.

Key results:

In cultured autaptic hippocampal neurons, JWH018 potently inhibited excitatory postsynaptic currents (IC₅₀= 14.9 nM) in a concentration- and CB₁ receptor-dependent manner. Furthermore, it increased ERK1/2 MAPK phosphorylation (EC₅₀= 4.4 nM). We also found that JWH018 potently induced rapid and robust CB₁ receptor internalization (EC₅₀= 2.8 nM; t_1/2= 17.3 min).

Conclusions and implications:

JWH018, a prominent component of several herbal preparations marketed for their psychoactivity, is a potent and effective CB₁ receptor agonist that activates multiple CB₁ receptor signalling pathways. Thus, it is likely that the subjective effects of ‘Spice’ are due to activation of cannabinoid CB₁ receptors by JWH018, added to this herbal preparation.     

Design,synthesis and biological evaluation of bivalent ligands against A1–D1 receptor heteromers

Aim:

To design and synthesize bivalent ligands for adenosine A₁–dopamine D₁ receptor heteromers (A₁–D₁R), and evaluate their pharmacological activities.

Methods:

Bivalent ligands and their corresponding A₁R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A₁R and D₁R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A₁–D₁R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D₁-CFP and A₁-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions.

Results:

Two bivalent ligands for A₁R and D₁R (20a, 20b), as well as the corresponding A₁R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A₁R 10–100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D₁R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D₁R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands.

Conclusion:

This study demonstrates the existence of A₁–D₁R in situ and a simultaneous interaction of bivalent ligands with both the receptors.     

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

BACKGROUND AND PURPOSE

The angiotensin II type 1 receptor (AT₁R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT₁R signals in a Gαq/11-dependent or -independent manner.

EXPERIMENTAL APPROACH

We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT₁R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts.

KEY RESULTS

Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p.

CONCLUSIONS AND IMPLICATIONS

Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells.     

Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

Background and Purpose

We previously reported that adenosine, acting at adenosine A_2A receptors (A_2AR), inhibits osteoclast (OC) differentiation in vitro (A_2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A_2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A_2AR signalling.

Experimental Approach

OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, the A_2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.

Key Results

A_2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A_2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A_2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A_2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.

Conclusions and Implications

A_2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.     

Physical and functional interaction between CB1 cannabinoid receptors and ��2-adrenoceptors

Background and purpose:

The CB₁ cannabinoid receptor and the β₂-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Experimental approach:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.

Key results:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β₂-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁ receptors. Co-expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i-dependent ERK phosphorylation, but decreased non-Gα_i-mediated CREB phosphorylation. The CB₁ receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β₂-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB₁ receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB₁ receptors and β₂-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β₂-adrenoceptor–pERK response.

Conclusion and implications:

A complex interaction was demonstrated between CB₁ receptors and β₂-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Levosimendan induces NO production through p38 MAPK,ERK and Akt in porcine coronary endothelial cells: role for mitochondrial KATP channel

Background and purpose:

Levosimendan acts as a vasodilator through the opening of ATP-sensitive K⁺ channels (K_ATP) channels. Moreover, the coronary vasodilatation caused by levosimendan in anaesthetized pigs has recently been found to be abolished by the nitric oxide synthase (NOS) inhibitor N^ω-nitro-L-arginine methyl ester, indicating that nitric oxide (NO) has a role in the vascular effects of levosimendan. However, the intracellular pathway leading to NO production caused by levosimendan has not yet been investigated. Thus, the purpose of the present study was to examine the effects of levosimendan on NO production and to evaluate the intracellular signalling pathway involved.

Experimental approach:

In porcine coronary endothelial cells (CEC), the release of NO in response to levosimendan was examined in the presence and absence of N^ω-nitro-L-arginine methyl ester, an adenylyl cyclase inhibitor, K_ATP channel agonists and antagonists, and inhibitors of intracellular protein kinases. In addition, the role of Akt, ERK, p38 and eNOS was investigated through Western blot analysis.

Key results:

Levosimendan caused a concentration-dependent and K⁺-related increase of NO production. This effect was amplified by the mitochondrial K_ATP channel agonist, but not by the selective plasma membrane K_ATP channel agonist. The response of CEC to levosimendan was prevented by the K_ATP channel blockers, the adenylyl cyclase inhibitor and the Akt, ERK, p38 inhibitors. Western blot analysis showed that phosphorylation of the above kinases lead to eNOS activation.

Conclusions and implications:

In CEC levosimendan induced eNOS-dependent NO production through Akt, ERK and p38. This intracellular pathway is associated with the opening of mitochondrial K_ATP channels and involves cAMP.     

Adenosine A1 receptor agonist N6-cyclohexyladenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

Aim:

To define the effect of adenosine A₁ receptor (A₁R) on delta opioid receptor (DOR)-mediated signal transduction.

Methods:

CHO cells stably expressing HA-tagged A₁R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A₁R agonist N⁶-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay.

Results:

CHA 1 μmol/L time-dependently attenuated DOR agonist [D-Pen^2,5]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t_1/2=2.56 (2.09–3.31) h. Pretreatment with 1 μmol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC₅₀ but no change in E_max. Pretreatment with 1 μmol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A₁R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K_d) nor receptor density (B_max) of DOR showed significant change after chronic CHA exposure.

Conclusion:

Activation of A₁R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A₁R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR.     

Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit

Aim:

Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BK_Ca channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BK_Ca channels.

Methods:

Human BK_Ca channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BK_Ca currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BK_Ca subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.

Results:

DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BK_Ca channels in HEK293 cells, associated with enhancements of Ca²⁺ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC₅₀ for BK_Ca channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.

Conclusion:

DS-201 selectively targets the pore-forming α subunit of human BK_Ca channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.     

Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

Background:

The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons.

Methods:

Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A_2A receptor modulation on intrinsic excitability.

Results:

Activation of adenosine A_2A receptors with the selective A_2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A_2A receptor-mediated effects were blocked by preapplication of a selective A_2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A_2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A_2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons.

Conclusions:

Collectively, these data suggest that adenosine, via activation of A_2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A₁ receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells.     

Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE

Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study.

EXPERIMENTAL APPROACH

We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed.

KEY RESULTS

BA inhibited LPS-induced COX-2 protein expression and prostaglandin E₂ production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE₂ production and myeloperoxidase activity in vivo.

CONCLUSIONS AND IMPLICATIONS

BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA.     

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/threonine kinases to activate ERK in neuronal cells

BACKGROUND AND PURPOSE

Signalling networks that regulate the progression of cannabinoid CB₁ receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB₁ receptor-stimulated ERK phosphorylation in a neuronal cell model.

EXPERIMENTAL APPROACH

Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB₁ receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.

KEY RESULTS

The time-course of CB₁ receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB₁ receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G_i/oβγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB₁ receptor-stimulated, ligand-independent, transactivation of multiple RTKs.

CONCLUSIONS AND IMPLICATIONS

The complex expression of CB₁ receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB₁-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng

BACKGROUND AND PURPOSE

Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms.

EXPERIMENTAL APPROACH

Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y₁₂ receptor was determined by molecular docking analysis, cytosolic Ca²⁺ and cAMP measurements, and phosphorylation of PI3K and Akt assays.

KEY RESULTS

Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca²⁺ accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y₁₂ receptors. The increase in intracellular Ca²⁺ evoked by Ft1 in HEK293 cells overexpressing P2Y₁₂ receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y₁₂ signalling pathways.

CONCLUSION AND IMPLICATIONS

Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y₁₂ receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.     

Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells

Background and purpose:

Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored.

Experimental approach:

RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5–8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE₂ and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-κB was analysed by Western blot; nuclear translocation of NF-κB was observed by immunofluorescence.

Key results:

Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE₂ and sICAM-1 levels. These effects were abolished by the CRF₂ receptor antagonist, antisauvagine-30, but not by the CRF₁ receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-κB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process.

Conclusions and implications:

These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF₂ receptors and the activation of p38MAPK and NF-κB.     

Mice lacking the Raf-1 kinase inhibitor protein exhibit exaggerated hypoxia-induced pulmonary hypertension

BACKGROUND AND PURPOSE

Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts.

EXPERIMENTAL APPROACH

Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP−/−) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts.

KEY RESULTS

RKIP−/− mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP−/− mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP−/− mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1.

CONCLUSIONS AND IMPLICATIONS

The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.     

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE

Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I_f or I_h). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels.

EXPERIMENTAL APPROACH

HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings.

KEY RESULTS

In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I_f by 67% at −120 mV, while MEL57A (3 µM) reduced I_f by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I_h by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization.

CONCLUSIONS

Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.     

β-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy

Aim:

To explore the effects of β-asarone from Acorus Tatarinowii Schott on autophagy in an ischemic stroke model of PC12 cells.

Methods:

The ischemic stroke model of PC12 cells was made by OGD/R (2 h oxygen-glucose deprivation followed by 24 h reperfusion). Drug administration was started 1 h before OGD and last for 3 h. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 24 h. After the treatments, Beclin-1, intracellular free calcium concentration ([Ca²⁺]_i) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Cell viability was measured using MTT assay. Cell morphology was studied under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope.

Results:

Pretreatment with β-asarone (20, 30, or 45 μg/mL) or the calcium channel antagonist nimodipine (10 μmol/L) significantly increased the cell viability and MMP, and decreased Beclin-1 expression and [Ca²⁺]_i in OGD/R-treated PC12 cells. Under inverted phase contrast microscope, pretreatment with β-asarone or nimodipine dramatically increase the number of cells and improved the cellular morphology. Autophagosomes were found in OGD/R-treated PC12 cells as well as in drug plus OGD/R-treated PC12 cells.

Conclusion:

β-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin-1-dependent autophagy caused by decreasing [Ca²⁺]_i and increasing MMP.     

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells

Aim:

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.

Methods:

HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.

Results:

In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.

Conclusion:

Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.