Inhibition Of Adenylate Cyclase Research Articles

Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors.

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure.

Inhibitory Effects of Rice Bran Water Extract Fermented Lactobacillus plantarum due to cAMP-dependent Phosphorylation of VASP (Ser157) on human Platelet Aggregation

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP (20 μM)-, collagen (10 μg/mL)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with IC50 values of 501.1, 637.2, and > 2,000 μg/mL, respectively. The platelet aggregation induced by ADP plus RBLp (750 μg/mL) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP (Ser 157 ), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP (Ser 157 ), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP (Ser 157 ). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

FNDC5 overexpression and irisin ameliorate glucose/lipid metabolic derangements and enhance lipolysis in obesity

Irisin is a cleaved and secreted fragment of fibronectin type III domain containing 5 (FNDC5), and contributes to the beneficial effects of exercise on metabolism. Here we report the therapeutical effects of FNDC5/irisin on metabolic derangements and insulin resistance in obesity, and show the lipolysis effect of irisin and its signal molecular mechanism. In obese mice, lentivirus mediated-FNDC5 overexpression enhanced energy expenditure, lipolysis and insulin sensitivity, and reduced hyperlipidemia, hyperglycemia, hyperinsulinism, blood pressure and norepinephrine levels; it increased hormone-sensitive lipase (HSL) expression and phosphorylation, and reduced perilipin level and adipocyte diameter in adipose tissues. Subcutaneous perfusion of irisin reduced hyperlipidemia and hyperglycemia, and improved insulin resistance. Either FNDC5 overexpression or irisin perfusion only induced a tendency toward a slight decrease in body weight in obese mice. In 3T3-L1 adipocytes, irisin enhanced basal lipolysis rather than isoproterenol-induced lipolysis, which were prevented by inhibition of adenylate cyclase or PKA; irisin increased the HSL and perilipin phosphorylation; it increased PKA activity, and cAMP and HSL mRNA levels, but reduced perilipin expression. These results indicate that FNDC5/irisin ameliorates glucose/lipid metabolic derangements and insulin resistance in obese mice, and enhances lipolysis via cAMP–PKA–HSL/perilipin pathway. FNDC5 or irisin can be taken as an effective therapeutic strategy for metabolic disorders.

Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions.

Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O2. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.

The traditional herbal medicine, Ge-Gen-Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP-sensitive K+ channels in cultured interstitial cells of Cajal from mouse small intestine

Ethnopharmacological relevanceGe-Gen-Tang (GGT) is a traditional Chinese medicinal formula composed of Puerariae radix (Pueraria lobata Ohwi), Ephedrae Herba (Ephedra sinica Stapf), Cinnamomi Ramulus (Cinnamomum cassia Blume), Paeoniae Radix (Paeonia lactiflora Pallas), Glycyrrhizae Radix preparata (Glycyrrhiza uralensis Fischer), Zingiberis Rhizoma (Zingiber officinale Roscoe), and Zizyphi Fructus (Ziziphus jujuba Mill. var. inermis Rehder) and is widely used to ameoliorate the symptoms of gastrointestinal (GI) disorders related to diarrhea and intestinal mucosal immunity and for anti-cold, antipyretic and analgesic in Eastern Asia. Aim of the studyInterstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. We investigated the effects of GGT on pacemaker potentials in cultured ICCs from the mouse small intestine, and sought to identify the receptors and the action mechanisms involved. Materials and methodsEnzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed on within 12h after culture. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. Intracellular Ca2+ ([Ca2+]i) increase was studied in cultured ICCs using fura-2AM. All of the experiments were performed at 30–32°C. ResultsUnder the current clamping mode, GGT decreased the amplitude and frequency of pacemaker potentials; however, these effects were blocked by intracellular GDPβS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K+ channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (β2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (β1-adrenoceptor antagonist) blocked the GGT-induced effects. Also, yohimbine (α2-adrenoceptor antagonist) partially blocked the GGT-induced effects. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the GGT-induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase, did. Additionally, [Ca2+]i analysis showed that GGT decreased [Ca2+]i. ConclusionThese results suggest that GGT inhibits pacemaker potentials in ICCs in a G protein-, cGMP- and NO-dependent manner through stimulation of α2 and β1-adrenoceptors.

Salusin-β contributes to vascular remodeling associated with hypertension via promoting vascular smooth muscle cell proliferation and vascular fibrosis

Vascular smooth muscle cell (VSMC) proliferation and vascular fibrosis are closely linked with hypertension and atherosclerosis. Salusin-β is a bioactive peptide involved in the pathogenesis of atherosclerosis. However, it is still largely undefined whether salusin-β is a potential candidate in the VSMC proliferation and vascular fibrosis. Experiments were carried out in human vascular smooth muscle cells (VSMCs) and in rats with intravenous injection of lentivirus expressing salusin-β. In vitro, salusin-β promoted VSMCs proliferation, which was attenuated by adenylate cyclase inhibitor SQ22536, PKA inhibitor Rp-cAMP, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, ERK inhibitor U0126 or cAMP response element binding protein (CREB) inhibitor KG501. It promoted the phosphorylation of ERK1/2, CREB and EGFR, which were abolished by SQ22536 or Rp-cAMP. Furthermore, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 diminished the salusin-β-evoked ERK1/2 and CREB phosphorylation. On the other hand, salusin-β increased collagen-I, collagen-III, fibronectin and connective tissue growth factor (CTGF) mRNA and phosphorylation of Smad2/3, which were prevented by ALK5 inhibitor A83-01. In vivo, salusin-β overexpression increased the media thickness, media/lumen ratio coupled with ERK1/2, CREB, EGFR and Smad2/3 phosphorylation, as well as the mRNA of collagen-I, collagen-III, fibronectin, transforming growth factor-β1 (TGF-β1) and CTGF in arteries. Moreover, salusin-β overexpression in rats caused severe hypertension. Intravenous injection of salusin-β dose-relatedly increased blood pressure, but excessive salusin-β decreased blood pressure and heart rate. These results indicate that salusin-β promotes VSMC proliferation via cAMP-PKA-EGFR-CREB/ERK pathway and vascular fibrosis via TGF-β1-Smad pathway. Increased salusin-β contributes to vascular remodeling and hypertension.

Expression and function of purinergic P2Y12 receptors in rat trigeminal ganglion neurons

Purinergic receptors play key signaling roles in neuropathic pain in the orofacial region, which is innervated by trigeminal ganglion (TG) neurons. The neuropathology of purinergic P2Y12 receptors is well characterized in glia; however, their physiological role in TG neurons remains to be fully elucidated. The present study investigated the expression and function of P2Y12 receptors in rat TG neurons. P2Y12 receptor immunoreactivity was intense in the soma, dendrites, and axons, and colocalized with a pan-neuronal marker, neurofilament H, isolectin B4, and substance P. In the presence of extracellular Ca2+, 2-methylthio-ADP (an agonist of P2Y1, 12, 13 receptors) transiently increased intracellular free Ca2+ concentrations ([Ca2+]i), an effect that was abolished by P2Y12 receptor antagonists. In the absence of extracellular Ca2+, ryanodine receptor/channel inhibitors diminished the 2-methylthio-ADP-induced increases in [Ca2+]i. A sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor gradually increased [Ca2+]i, and after a plateau, application of 2-MeS-ADP induced a rapid and transient, but additive increase in [Ca2+]i. An adenylate cyclase inhibitor transiently increased [Ca2+]i, while a phosphodiesterase inhibitor prevented the 2-methylthio-ADP-induced increase in [Ca2+]i. Our study shows that P2Y12 receptors are expressed in TG neurons, and act via a cAMP-dependent pathway to release intracellular Ca2+ from ryanodine-sensitive Ca2+ stores.

Increased β2-adrenergic vasorelaxation at the early phase of endotoxemic shock in rats

Background and purposeThe early management of the cardiovascular dysfunction of septic shock is critical as it is associated with a poor outcome. Although the use of catecholamines is a common therapy in this syndrome, no data are available on the involvement of β-adrenoceptor (β-AR) subtypes and only few studies report an alteration of β-adrenergic-induced vasodilation in septic shock. The purpose of the study was to evaluate vascular β1, β2 and β3-AR expression and function in an endotoxemic rat model. Experimental approachEndotoxemia was induced in rats by intravenous injection of lipopolysaccharide (LPS). β1, β2 and β3-AR mRNA expression was evaluated by RT-PCR in aorta and vascular β1, β2 and β3-AR responses were determined on conducting (aorta) and/or resistance (mesenteric and renal) arteries by constructing relaxation curves in response to different β-AR agonists. ResultsThe maximal effect of isoproterenol decreased by 31 to 61% in the three vascular beds of LPS-treated rats compared to controls. In aortas from LPS-treated rats, β1 and β3-AR mRNA expression was decreased and associated to a reduced β1 and β3-induced vasodilation. Conversely, albeit β2-AR mRNA was unchanged, the maximal β2-AR-induced vasodilation increased by 49% in aortas from LPS-treated rats compared to controls. This increase was not affected by endothelium removal but was abolished in the presence of a β2-AR antagonist or an adenylate cyclase inhibitor. ConclusionsIn endotoxemia, β2-AR vasodilation was increased by a potential recruitment of β2-AR located on smooth muscle cells. This study suggests that vascular β2-AR should be a putative new therapeutic target in septic shock.

Facilitation of corticostriatal transmission following pharmacological inhibition of striatal phosphodiesterase 10A: role of nitric oxide-soluble guanylyl cyclase-cGMP signaling pathways.

The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade.

Regulation of Novel Calcium‐Sensitive Dicarboxylate Transport by the Calcium‐Sensing Receptor

Citrate inhibits calcium nephrolithiasis by complexing calcium (Ca) in a soluble form. Lowering apical extracellular Ca from 1.2mM (normal levels, Nl) to <60μM (low) results in an increase in Cit and succinate (Suc) transport in OK cells. Activation of the calcium-sensing receptor (CaSR) with spermine inhibits transport in both low and Nl Ca. But activation of PKC with PMA inhibited transport in low Ca only, implying that Ca-sensitive dicarboxylate transport is regulated by the CaSR through the Gq/PKC pathway. The purpose here is to determine the role of the Gi pathway. 14C-Suc uptakes were performed in OK cells and HRPE cells transfected with NaDC1 (CUBS). CaSRs inhibit adenylate cyclase (AC) through Gi. To mimic Gi, cells were pre-incubated with an AC inhibitor, MDL-12,330A (50μM for 30min) followed by uptake. The addition of MDL inhibited uptake in both Nl (0.09±0.01 to 0.05±0.01, p<0.01) and low Ca (0.13±0.01 to 0.08±0.01, p<0.01). In CUBS uptake was also inhibited in Nl (0.6±0.02 to 0.3±0.03, p<0.01) and low Ca (0.5±0.04 to 0.2±0.03, p<0.01). These results are similar to our studies of CaSR activation by spermine. If MDL inhibits transport via AC inhibition, 8-Br-cAMP (8-Br) should reverse MDL action. In OK cells, MDL eliminated the Ca-sensitivity; i.e. Nl (0.05±0.004) vs low Ca (0.05±0.002). In low Ca, 8-Br negated the inhibitory effects of MDL thus significantly increasing uptake (0.049±0.002 to 0.063±0.002, p<0.05). Thus stimulation of the CaSR through both Gq and Gi indicates CaSR involvement in the regulation of Ca-sensitive citrate transport. NIH/NIDDK

ASM-024, a piperazinium compound, promotes the in vitro relaxation of β2-adrenoreceptor desensitized tracheas.

Inhaled β2-adrenoreceptor agonists are widely used in asthma and chronic obstructive pulmonary disease (COPD) for bronchoconstriction relief. β2-adrenoreceptor agonists relax airway smooth muscle cells via cyclic adenosine monophosphate (cAMP) mediated pathways. However, prolonged stimulation induces functional desensitization of the β2-adrenoreceptors (β2-AR), potentially leading to reduced clinical efficacy with chronic or prolonged administration. ASM-024, a small synthetic molecule in clinical stage development, has shown activity at the level of nicotinic receptors and possibly at the muscarinic level and presents anti-inflammatory and bronchodilator properties. Aerosolized ASM-024 reduces airway resistance in mice and promotes in-vitro relaxation of tracheal and bronchial preparations from animal and human tissues. ASM-024 increased in vitro relaxation response to maximally effective concentration of short—acting beta-2 agonists in dog and human bronchi. Although the precise mechanisms by which ASM-024 promotes airway smooth muscle (ASM) relaxation remain unclear, we hypothesized that ASM-024 will attenuate and/or abrogate agonist-induced contraction and remain effective despite β2-AR tachyphylaxis. β2-AR tachyphylaxis was induced with salbutamol, salmeterol and formoterol on guinea pig tracheas. The addition of ASM-024 relaxed concentration-dependently intact or β2-AR desensitized tracheal rings precontracted with methacholine. ASM-024 did not induce any elevation of intracellular cAMP in isolated smooth muscle cells; moreover, blockade of the cAMP pathway with an adenylate cyclase inhibitor had no significant effect on ASM-024-induced guinea pig trachea relaxation. Collectively, these findings show that ASM-024 elicits relaxation of β2-AR desensitized tracheal preparations and suggest that ASM-024 mediates smooth muscle relaxation through a different target and signaling pathway than β2-adrenergic receptor agonists. These findings suggest ASM-024 could potentially provide clinical benefit when used adjunctively with inhaled β2-adrenoreceptor agonists in those patients exhibiting a reduced response to their chronic use.

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]i and then potentiate glucose-induced insulin secretion and production in parallel.

Inhibitory Mechanisms of CME-1, a Novel Polysaccharide from the Mycelia of Cordyceps sinensis, in Platelet Activation

CME-1 is a polysaccharide purified from the mycelia of medicinal mushroom Cordyceps sinensis, its molecular weight was determined to be 27.6 kDa by using nuclear magnetic resonance and gas chromatography-mass spectrometry. The initiation of arterial thromboses is relevant to various cardiovascular diseases (CVDs) and is believed to involve platelet activation. Our recent study exhibited that CME-1 has potent antiplatelet activity via the activation of adenylate cyclase/cyclic AMP ex vivo and in vivo. The aggregometry, and immunoblotting were used in this study. In this study, the mechanisms of CME-1 in platelet activation is further investigated and found that CME-1 inhibited platelet aggregation as well as the ATP-release reaction, relative intracellular [Ca(+2)] mobilization, and the phosphorylation of phospholipase C (PLC)γ2 and protein kinase C (PKC) stimulated by collagen. CME-1 has no effects on inhibiting either convulxin, an agonist of glycoprotein VI, or aggretin, an agonist of integrin α2β1 stimulated platelet aggregation. Moreover, this compound markedly diminished thrombin and arachidonic acid (AA) induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2, c-Jun N-terminal kinase 1, and Akt. Treatment with SQ22536, an inhibitor of adenylate cyclase, markedly diminished the CME-1-mediated increasing of cyclic AMP level and reversed prostaglandin E1- or CME-1-mediated inhibition of platelet aggregation and p38 MAPK and Akt phosphorylation stimulated by thrombin or AA. Furthermore, phosphodiesterase activity of human platelets was not altered by CME-1. The crucial finding of this study is that the antiplatelet activity of CME-1 may initially inhibit the PLCγ2-PKC-p47 cascade, and inhibit PI3-kinase/Akt and MAPK phosphorylation through adenylate cyclase/ cyclic AMP activation, then inhibit intracellular [Ca(+2)] mobilization, and, ultimately, inhibit platelet activation. The novel role of CME-1 in antiplatelet activity indicates that this compound exhibits high therapeutic potential for treating or preventing CVDs.

NaHS induces relaxation response in prostaglandin F2α precontracted bovine retinal arteries partially via Kv and Kir channels

Hydrogen sulphide (H2S) is known to be produced endogenously in ocular tissues with the highest levels in the retina and cornea. However, it is yet unclear whether it can modulate retinal arterial tone. Herein, we aimed to investigate the effectiveness and the mechanism of the action of H2S in the isolated bovine retinal arteries. For this purpose, the probable vasorelaxant and inhibitory effects of H2S on vascular reactivity were tested comparatively in the retinal arteries by using the donor, sodium hydrosulphide (NaHS). Thereafter, in relation to the mechanism of action of H2S, the role of nitric oxide (NO) and endothelial vasodilators of cyclooxygenase pathway as well as ATP-sensitive potassium channel (KATP), voltage-dependent potassium channel (Kv), calcium-activated potassium channel (KCa++), inwardly rectifying potassium channel (Kir), L-type voltage-dependent calcium channel and adenylate cyclase pathway were evaluated. NaHS (1μM–3mM) displayed prominent relaxations over the concentrations of 300 μM in both PGF2α and K+ precontracted retinal arteries. Comparatively, in the presence of NaHS (3 mM) pretreatment, the maximum contractile responses and pEC50 values to PGF2α and K+ were significantly reduced as well. Neither the presence of the known inhibitors of NO synthase, guanylate cyclase, cyclooxygenase, adenylate cyclase, KATP and KCa++ type K+ channels, and L-type voltage-dependent calcium channels nor the removal of endothelium, modified the relaxation response to NaHS in retinal arteries. However, a remarkable decrease was observed in the presence of the inhibitors of Kv or Kir type K+ channels. In addition, administration of l-cysteine (1μM–3mM), the precursor of H2S, induced a modest relaxation response in PGF2α precontracted retinal arteries, which was significantly decreased in the presence of cystathionine-β-synthase (CBS) inhibitor, aminooxyacetic acid, but was unmodified in the presence of the cystathionine-γ-lyase (CSE) inhibitor, dl-propargylglycine or the deendothelization of retinal arteries. Our findings suggested that H2S might play a substantial role in the regulation of retinal arterial tone possibly by acting on Kv and Kir channels.

Cell entry of bovine ephemeral fever virus requires activation of Src-JNK-AP1 and PI3K-Akt-NF-κB pathways as well as Cox-2-mediated PGE2 /EP receptor signalling to enhance clathrin-mediated virus endocytosis.

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin-mediated and dynamin 2-dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src-JNK-AP1 and PI3K-Akt-NF-κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src-JNK signalling to enhance its entry. Activation of these pathways by ultraviolet-inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV-induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3'-dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox-2-catalysed prostaglandin E2 (PGE2) synthesis and induces expressions of G-protein-coupled E-prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src-JNK-AP1 and PI3K-Akt-NF-κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV-induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox-2-mediated PGE2/EP receptor signalling pathways, further enhancing Src-JNK-AP1 in a cAMP-dependent manner and PI3K-Akt-NF-κB in a cAMP-independent manner. Accordingly, BEFV stimulates PGE2/EP receptor signalling amplifying Src-JNK-AP1 and PI3K-Akt-NF-κB pathways in an autocrine or paracrine fashion to enhance virus entry.

The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation.

PTHrP regulates water absorption and aquaporin expression in the intestine of the marine sea bream (Sparus aurata, L.)

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3− secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3− secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3− secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3− secretion rapidly decreased when PTHrP(1–34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1ngml−1 compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100μmoll−1) or the phospholipase C inhibitor (U73122, 10μmoll−1) the effect of PTHrP(1–34) on HCO3− secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose–response curve in the range of 0.1–1000ngml−1, achieves a maximal effect of 60–65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1ngml−1. The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100μmoll−1) and was insensitive to the phospholipase C inhibitor (U73122, 10μmoll−1). In vivo injections of PTHrP(1–34) or the PTH/PTHrP receptor antagonist PTHrP(7–34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO3− secretion.

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal.

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K+ channel blocker). However, neither NG-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K+ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (I_SC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The I_SC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca²⁺]_i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in I_SC, which was due to Cl^- secretion. The increase in I_SC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTR_inh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated I_SC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in I_SC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca²⁺]_i. Conclusion: Nobiletin stimulated transepithelial Cl^- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl^- channels.