Epinephrine activates both Gs and Gi pathways, but norepinephrine activates only the Gs pathway through human beta2-adrenoceptors overexpressed in mouse heart

Isoproterenol increases and decreases contractile force at low and high concentrations, respectively, through beta(2)-adrenoceptors overexpressed in transgenic mouse heart (TG4), consistent with activation of both G(s) and G(i) proteins. Using TG4 hearts, we demonstrated that epinephrine behaves like isoproterenol, but norepinephrine does not. Epinephrine both increased (-log EC(50)M = 9.4) and decreased (-log EC(50)M = 6.5) left atrial force. Pertussis toxin (PTX) abolished the negative inotropic effects of epinephrine, consistent with mediation through G(i) protein. Norepinephrine only increased contractile force (-log EC(50)M = 7.5). Norepinephrine (10-100 microM) prevented the positive inotropic effects but hardly affected the negative inotropic effects of epinephrine. Cardiodepressive epinephrine concentrations (1-10 microM) antagonized the positive inotropic effects of norepinephrine. In the free wall of TG4 right ventricle, norepinephrine and low epinephrine concentrations caused positive inotropic effects, and high epinephrine concentrations caused PTX-sensitive negative inotropic effects, as observed in the left atrium. Epinephrine (10 nM), a concentration causing maximum increase in contractile force, and norepinephrine (1 and 100 microM) increased cAMP-dependent protein kinase activity in TG4 left ventricle. Cardiodepressive concentrations of epinephrine (1 and 100 microM) did not increase cAMP-dependent protein kinase activity. The inotropic results were simulated with a model of two beta(2)-adrenoceptor sites. For one site involved in receptor coupling to G(s), both epinephrine and norepinephrine compete. The other site, recognized by epinephrine but not by norepinephrine, leads to receptor G(i) coupling.     

The relaxin family peptide receptor 3 activates extracellular signal-regulated kinase 1/2 through a protein kinase C-dependent mechanism

Human gene 3 relaxin (H3 relaxin) is a member of the relaxin/insulin family of peptides. Neuropeptides mediate behavioral responses to stress and regulates appetite; however, the cell signaling mechanisms that control these events remain to be identified. The relaxin family peptide receptor 3 (RXFP3, formerly GPCR135 or SALPR) was characterized as the receptor for H3 relaxin, functionally coupled to the inhibition of cAMP. We have identified that RXFP3 stably expressed in Chinese hamster ovary (CHO)-K1 (CHO-RXFP3) and human embryonic kidney (HEK) 293 (HEK-RXFP3) cells activates extracellular signal-regulated kinase (ERK) 1/2 when stimulated with H3 relaxin and an H3 relaxin B-chain (dimer) peptide. Using inhibitors of cellular signaling proteins, we subsequently determined the mechanism of ERK1/2 activation by RXFP3. ERK1/2 phosphorylation requires the activation of G(i/o) proteins and seems to require receptor internalization and/or compartmentalization into lipid-rich environments. ERK1/2 activation also predominantly occurred via the activation of a protein kinase C-dependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase were also involved to a lesser extent. The mechanisms underlying ERK1/2 phosphorylation were similar in both CHO-RXFP3 and HEK-RXFP3 cells, although some differences were evident. Phospholipase Cbeta and the transactivation of endogenous epidermal growth factor receptors both played a role in RXFP3-mediated ERK1/2 activation in HEK293 cells; however, they were not involved in RXFP3-mediated ERK1/2 activation in the CHO-K1 cell background. The pathways identified in CHO- and HEK-transfected cells were also used in the murine SN56 neuronal cell line, suggesting that these pathways are also important for RXFP3-mediated signaling in the brain.     

Curdlan activates dendritic cells through dectin-1 and toll-like receptor 4 signaling

Curdlan, a β-1,3-glucan isolated from Alcaligenes faecalis, is an agonist of dectin-1 in various immune cells, including dendritic cells (DCs). However, whether curdlan also activates DCs through other receptors remains unknown. In this study, we found that curdlan activates DCs through dectin-1 and toll-like receptor 4 (TLR4). Curdlan increased the expression levels of surface molecules (CD40, CD80, CD86, and MHC-I/II), the production of cytokines (IL-12, IL-1β, TNF-α, and IFN-β), migration toward MIP-3β, and allogeneic T cell stimulation activity of DCs. Curdlan increased the phosphorylation of Syk, Raf-1, Akt, MAPKs, IKK, and NF-κB p65 in DCs. However, curdlan only slightly activated DCs transfected with small interfering RNAs against dectin-1 or TLR4 and C3H/HeJ DCs, which have non-functional TLR4, in comparison with control DCs. Curdlan increased antitumor activity of DCs in a syngeneic tumor model. In summary, our data show that curdlan activates DCs through dectin-1 and TLR4 signaling and the combination of curdlan and DCs efficiently inhibit tumor growth in mice.     

The diacylglycerol kinase inhibitor, R59949, potentiates secretion but not increased phosphorylation of a 47 kDalton protein in human platelets.

We have investigated the action of a novel inhibitor of DG-kinase, R59949. This agent was found to produce partial inhibition of formation of phosphatidic acid in human platelets challenged with thrombin, DC8 or OAG. However, this effect was not associated with enhanced phosphorylation of a 47 kDa protein, a known substrate for protein kinase C. We therefore believe that this compound does not represent a major advance on its earlier prototype, R59022.     

Perazine as a potent inhibitor of human CYP1A2 but not CYP3A4

The effects of perazine on the activities of CYP1A2 and CYP3A4 in a primary culture of human hepatocytes of one patient were studied in vitro. The CYPs activities were assessed by measuring the rate of acetanilide 4-hydroxylation (CYP1A2) and cyclosporine A oxidation (CYP3A4) after treatment with TCDD (a CYP1A subfamily inducer) or rifampicin (mainly a CYP3A4 inducer). The amounts of the metabolites formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of 4-hydroxyacetanilide and cyclosporine A metabolites (monohydroxycyclosporine A, dihydroxycyclosporine A, N-desmethylcyclosporine A), respectively. The formation of 4-hydroxyacetanilide was strongly inhibited by three different concentrations of perazine (10, 25 and 50 microM) reaching 8, 3 and 2% of the control value, respectively. In the case of CYP3A4 activity, no such an effect of perazine was observed. Perazine showed only a week inhibition of the activity of cyclosporine A oxidase (to 96-86% of the control value). The obtained results suggest a strong inhibitory effect of perazine on human CYP1A2 activity with predicted Ki value similar to those of the known for CYP1A2 inhibitors, such as furafylline and fluvoxamine.     

Differential effects of quercetin,apigenin and genistein on signalling pathways of protease-activated receptors PAR1 and PAR4 in platelets

Background and purpose:

The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors.

Experimental approach:

We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using ¹²⁵I-thrombin.

Key results:

Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested.

Conclusions and implications:

Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.     

Alpha-lipoic acid activates eNOS through activation of PI3-kinase/Akt signaling pathway

BackgroundLipoic acid (LA) exerts therapeutic effects on cardiovascular diseases. However, the mechanisms underlying these therapeutic effects remain elusive. Endothelial nitric oxide synthase (eNOS) plays a critical role in cardiovascular homeostasis. LA was shown to potently activate PI3-kinase/Akt pathway, and the latter is critical in the regulation of eNOS activity. In the present study, we test the hypothesis that LA improves endothelial function through PI₃-kinase/Akt-mediated eNOS activation.Methods and ResultsWestern blot analysis showed that LA time- and dose-dependently induced phosphorylation of Akt and eNOS in human umbilical vein endothelial cells (HUVECs). Both PI₃-kinase and Akt inhibitors abolished LA-induced eNOS phosphorylation, indicating that LA induces eNOS phosphorylation through the PI₃-kinase/Akt pathway. This increase in eNOS phosphorylation was paralleled by an increase in NO release by HUVECs, supporting its relevance in eNOS activity regulation. Myograph analysis revealed that LA relaxed phenylephrine-induced contraction. Endothelium removal and NOS inhibition by L-NAME abolished this vasodilator action of LA, and Akt but not AMPK inhibition significantly reduced the vasodilator action of LA, indicating that it is mediated by PI₃-kinase/Akt pathway-dependent activation of eNOS. Consistent with in vitro results, intraperitoneal injection with LA significantly increased plasma nitrite and nitrate levels in C57Bl/6j mice.ConclusionsLA activates eNOS through a PI3-kinase/Akt signaling pathway-dependent mechanism, offering a potential molecular basis for the therapeutic effects of LA on cardiovascular diseases.     

The frog trefoil factor Bm-TFF2 activates human platelets via Gq and G12/13 signaling pathway

Bm-TFF2 is an amphibian trefoil factor purified from the Bombina maxima skin secretion that is highly toxic to mammals. We previously reported that Bm-TFF2 activates human platelets via protease-activated receptor 1. In this study, for a better understanding of platelet activation induced by Bm-TFF2, we used affinity chromatography and pharmacological inhibitors to investigate the downstream signaling pathway. Using Bm-TFF2-affinity chromatography, Gq was specifically eluted from the Bm-TFF2-coulped column. Pharmacological inhibitors such as U73122, Xestospongin C, BAPTA-AM and Gö6976 can significantly inhibit Bm-TFF2-induced platelet aggregation. These results suggested that Gq activation and the downstream PLCβ-IP3 receptor-cytoplasmic Ca²⁺-PKC signaling pathway is crucial for Bm-TFF2 to stimulate platelet aggregation. Furthermore, Bm-TFF2 induced strong platelet shape change at the concentrations of 5 nM, in which the Ca²⁺ mobilization of the platelets stimulated was not detectable. The p160^ROCK inhibitorY27632 totally inhibited the shape change, indicating that Bm-TFF2 may activate the G12/13 pathway which leads to the activation of RhoA-p160^ROCK. In conclusion, Bm-TFF2 induced platelet activation mainly via the Gq and G12/13 signaling pathway. This study on the signaling pathway of Bm-TFF2 stimulation may help us understand the toxicity of B. maxima skin secretion to the human platelets.     

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins.Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.     

Protean agonism at the dopamine D2 receptor: (S)-3-(3-hydroxyphenyl)-N-propylpiperidine is an agonist for activation of Go1 but an antagonist/inverse agonist for Gi1,Gi2, and Gi3

A range of ligands displayed agonism at the long isoform of the human dopamine D(2) receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxin-resistant mutants of individual Galpha(i)-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [(35)S]GTPgammaS onto each of Galpha(i1),Galpha(i2),Galpha(i3), and Galpha(o1). By contrast, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when Galpha(o1) was the target G protein but an antagonist/inverse agonist at Galpha(i1),Galpha(i2), and Galpha(i3). In ligand binding assays, dopamine identified both high- and low-affinity states at each of the dopamine D(2) receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D(2) receptor was fused to Galpha(i1),Galpha(i2), or Galpha(i3). However, it bound to distinct high- and low-affinity states of the D(2) receptor-Galpha(o1) fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D(2) receptor when expression of pertussis toxin-resistant forms of each of Galpha(i1), Galpha(i2), Galpha(i3), and Galpha(o1) was induced, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of Galpha(o1). p-Tyramine displayed a protean ligand profile similar to that of (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D(2) receptor and may explain in vivo actions of this ligand.     

Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation

Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis.     

Phospholipase C-beta3 and -beta1 form homodimers, but not heterodimers, through catalytic and carboxyl-terminal domains

Phospholipase C-beta (PLC-beta) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-beta may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-beta constructs and size-exclusion chromatography of native PLC-beta, we observed homodimerization of PLC-beta3 and PLC-beta1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-beta3 and PLC-beta1 form higher affinity homodimers than PLC-beta2. Evidence supportive of limited PLC-beta monomer-homodimer equilibrium appears at < or =100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-beta3 fragments demonstrated that at least two subdomains of PLC-beta3 are involved in dimer formation, one in the catalytic X and Y domains and the other in the G protein-regulated carboxyl-terminal domain. In addition, we provide evidence consistent with the existence of PLC-beta homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.     

Palmatine activates AhR and upregulates CYP1A activity in HepG2 cells but not in human hepatocytes

The protoberberine alkaloid palmatine is present in preparations from medicinal plants such as Coptis chinensis and Corydalis yanhusuo. This study examined whether palmatine affects the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells grown as monolayer or spheroids. Gene reporter assays showed that palmatine significantly activated the aryl hydrocarbon receptor (AhR) and increased the activity of CYP1A1 gene promoter in transiently transfected HepG2 cells. In HepG2 monolayer culture, palmatine also significantly increased mRNA and activity levels of CYP1A1, albeit with considerably less potency than 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical CYP1A inducer. On the other hand, CYP1A activity was not significantly elevated by palmatine in HepG2 spheroids. Moreover, palmatine induced mild or negligible changes in CYP1A1 and CYP1A2 mRNA expression without affecting CYP1A activity levels in primary human hepatocytes. It is concluded that palmatine activates the AhR-CYP1A pathway in HepG2 monolayer, while the potential for CYP1A induction is irrelevant in cell systems which are closer to the in vivo situation, i.e. in HepG2 spheroids and primary cultures of human hepatocytes. Possible induction of CYP1A enzymes by palmatine in vivo remains to be investigated.     

The dopamine agonism on ADP-stimulated platelets is mediated through D2-like but not D1-like dopamine receptors

Monoamines such as serotonin and epinephrine are known to be involved in platelet activation and aggregation. Dopamine is another monoamine identified in platelets, but published data about its effect on platelets and the receptors involved are controversial. In the present study, we investigated the dopamine agonism in platelets and the receptors involved in these pathways. Platelet-rich plasma (PRP) of healthy individuals was treated with agonists (ADP, epinephrine, dopamine) and various dopamine receptor and transporter antagonists such as SCH-23390, raclopride, clozapine, methylphenidate, and cocaine. Platelet activation was investigated by flow cytometry (CD62P and CD63 surface expression), optical aggregometry, and microaggregate adhesion to collagen IV in a flow chamber system. In our study, dopamine on its own had no effect on platelet activation in the different assays. However, when used in combination with ADP (10 μM), dopamine in a range of 1 to 100 μM significantly potentiated platelet microaggregate formation and adhesion to collagen under low shear flow conditions. Specific antagonists for D2-like receptors (L-741,626, raclopride, and clozapine) completely diminished the dopamine effect at selective concentrations, but not the effect of epinephrine. Neither the D1-like receptor antagonist SCH-23390 nor dopamine transporter antagonists (methylphenidate, cocaine) showed inhibitory effects on the dopamine agonism. Thus, dopamine is an ADP-dependent platelet agonist which acts via D2-like but not D1-like receptors or adrenergic receptors. Because many psychopharmacological drugs are directed to D2-like receptors, platelet dysfunction in patients being treated with such drugs may be linked to these mechanisms.     

K252a prevents nigral dopaminergic cell death induced by 6-hydroxydopamine through inhibition of both mixed-lineage kinase 3/c-Jun NH2-terminal kinase 3 (JNK3) and apoptosis-inducing kinase 1/JNK3 signaling pathways

It is well documented that the mitogen-activated protein kinase pathway plays a pivotal role in rats with 6-hydroxydopamine (6-OHDA)-induced unilateral lesion in the nigrostriatal system. Our recent studies have shown that mixed-lineage kinase 3 (MLK3) and apoptosis-inducing kinase 1 (ASK1) are all involved in neuronal cell death induced by ischemia, which is mediated by the MLK3/c-Jun NH2-terminal kinase 3 (JNK3) and ASK1/JNK signaling pathway. To investigate whether these pathways are correlated with 6-OHDA-induced lesion as well, we examined the phosphorylation of MLK3, ASK1, and JNK3 in 6-OHDA rats. The results showed that both MLK3 and ASK1 could activate JNK3 and then subsequently enhance the neuronal death through its downstream pathways (i.e., nuclear and non-nuclear pathway). K252a have wide-range effects including Trk inhibition, MLK3 inhibition, and activation of phosphatidylinositol 3 kinase and mitogen-activated protein kinase kinase signaling pathways through interactions with distinct targets and is a well known neuroprotective compound. We found that K252a could protect dopaminergic neurons against cell program death induced by 6-OHDA lesion, and the phenotypes of 6-OHDA rat model treated with K252a were partial rescued. The inhibition of K252a on the activation of MLK3/JNK3 and ASK1/JNK3 provided a link between 6-OHDA lesion and stress-activated kinases. It suggested that both proapoptotic MLK3/JNK3 and ASK1/JNK3 cascade may play an important role in dopaminergic neuronal death in 6-OHDA insult. Thus, the JNK3 signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of Parkinson disease, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in Parkinson disease.     

Tandospirone activates neuroendocrine and ERK (MAP kinase) signaling pathways specifically through 5-HT1A receptor mechanisms in vivo

Tandospirone, an azapirone, is a selective serotonin_1A (5-HT_1A) receptor agonist. The effects of tandospirone on plasma hormones and on mitogen-activated protein (MAP) kinase activity in the brain of male rats were studied. Tandospirone produced a time- and dose-dependent increase in plasma levels of oxytocin, adrenocorticotropin (ACTH), corticosterone, and prolactin. The minimal dose of tandospirone that led to a significant elevation of plasma oxytocin, ACTH, and prolactin levels was 1.0 mg/kg (s.c.), while the minimal dose for corticosterone release was 3.0 mg/kg (s.c.). The ED₅₀ of tandospirone was 1.3 mg/kg for oxytocin, 1.2 mg/kg for ACTH, 3.0 mg/kg for corticosterone, and 0.24 mg/kg for prolactin. Pretreatment with the specific 5-HT_1A receptor antagonist WAY 100,635 (0.3 mg/kg, s.c.) completely blocked the effects of tandospirone on plasma levels of oxytocin, ACTH, and corticosterone but shifted the dose–response curve for prolactin to the right. Tandospirone injection (10 mg/kg, s.c.) stimulated the MAP kinase signaling cascade, specifically the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Western blot analysis revealed a significant increase in phosphorylated ERK (p-ERK) levels in the hypothalamic paraventricular nucleus (PVN) as well as the dorsal raphé nucleus 5 min following tandospirone injection. These increases were blocked by pretreatment with WAY 100,635 (0.3 mg/kg). The results are the first evidence that systemic 5-HT_1A receptor agonist administration produces a rapid increase in p-ERK levels in vivo, providing further insight into the signaling mechanisms of the 5-HT_1A receptor.     

Inhibition of c-Jun N-terminal kinase 1, but not c-Jun N-terminal kinase 2, suppresses apoptosis induced by ischemia/reoxygenation in rat cardiac myocytes

In the present study, rat cardiac myocytes were used as an in vitro ischemia/reperfusion injury model to delineate the role of c-Jun N-terminal kinase (JNK) 1 and JNK2 isoforms in ischemia/reoxygenation-induced apoptosis using an antisense approach. Exposure of rat cardiac myocytes to ischemia did not induce apoptosis as detected by staining with either acridine orange/ethidium bromide or annexin-V-fluorescein/propidium iodide. In contrast, a time-dependent increase in the number of apoptotic cells was noted after reoxygenation of ischemic myocytes, whereas the level of necrotic cells remained unaltered. Reoxygenation, but not ischemia alone, also caused a time-dependent increase in JNK activation that preceded apoptosis induction. Treatment of cardiac myocytes with antisense (AS) oligonucleotides that specifically targeted either JNK1 or JNK2 significantly reduced both mRNA and protein expression of the target isoform but had no effect on the expression of the alternate isoform. Pretreatment of cardiac myocytes with JNK1 AS, but not JNK2 AS, resulted in almost complete attenuation of reoxygenation-induced apoptosis. Furthermore, control oligonucleotides for JNK1 AS or JNK2 AS had no effect on JNK mRNA or protein expression or reoxygenation-induced apoptosis, indicating a sequence-specific mode of action. Additional studies revealed that apoptosis induced by other JNK-activating stimuli, including ceramide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS but not JNK2 AS. These findings demonstrate that the JNK1 isoform plays a preferential role in apoptosis induced by ischemia/reoxygenation as well as diverse JNK-activating cellular stresses.