Protein kinase A signalling is involved in the relaxant responses to the selective β-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro

Objectives The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium‐denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action. Methods cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. Key findings DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine‐precontracted aortic rings (10–300 µM). DPN‐induced vasorelaxation was largely reduced by the PKA inhibitors Rp‐8‐Br‐cAMPS (8‐bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer) and H‐89 (N‐(2‐bromocynnamyl(amino)ethyl)‐5‐isoquinoline sulfonamide HCl) (?73%) and by the adenylate cyclase inhibitor MDL12330A (cis‐N‐(2‐phenylcyclopentyl)‐azacyclotridec‐1‐en‐2‐amine)) (?65.5%). Conversely, the PKG inhibitor Rp‐8‐Br‐cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA‐dependent HSP20 phosphorylation, an effect reversed by H‐89. Relaxant responses to DPN were modestly antagonised (?23 to ?48% reduction; n = 12 per compound) by the potassium channel inhibitors iberiotoxin, PNU‐37883A, 4‐aminopyridine, or BaCl₂. All four potassium channel inhibitors together reduced DPN relaxation by 86 ± 9% (n = 12) and fully blocked DPN hyperpolarisation. Conclusions ERβ‐dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.     

The anti-diabetic drug repaglinide induces vasorelaxation via activation of PKA and PKG in aortic smooth muscle

We investigated the vasorelaxant effect of repaglinide and its related signaling pathways using phenylephrine (Phe)-induced pre-contracted aortic rings. Repaglinide induced vasorelaxation in a concentration-dependent manner. The repaglinide-induced vasorelaxation was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor (L-NAME) and a small-conductance Ca^2 +-activated K⁺ (SK_Ca) channel inhibitor (apamin) did not alter the vasorelaxant effect of repaglinide on endothelium-intact arteries. Pretreatment with an adenylyl cyclase inhibitor (SQ 22536) or a PKA inhibitor (KT 5720) effectively reduced repaglinide-induced vasorelaxation. Also, pretreatment with a guanylyl cyclase inhibitor (ODQ) or a PKG inhibitor (KT 5823) inhibited repaglinide-induced vasorelaxation. However, pretreatment with a voltage-dependent K⁺ (Kv) channel inhibitor (4-AP), ATP-sensitive K⁺ (K_ATP) channel inhibitor (glibenclamide), large-conductance Ca^2 +-activated K⁺ (BK_Ca) channel inhibitor (paxilline), or the inwardly rectifying K⁺ (Kir) channel inhibitor (Ba^2 +) did not affect the vasorelaxant effect of repaglinide. Furthermore, pretreatment with a Ca^2 + inhibitor (nifedipine) and a sarco-endoplasmic reticulum Ca^2 +-ATPase (SERCA) inhibitor (thapsigargin) did not affect the vasorelaxant effect of repaglinide. The vasorelaxant effect of repaglinide was not affected by elevated glucose (50 mM). Based on these results, we conclude that repaglinide induces vasorelaxation via activation of adenylyl cyclase/PKA and guanylyl cyclase/PKG signaling pathways independently of the endothelium, K⁺ channels, Ca^2 + channels, and intracellular Ca^2 + ([Ca^2 +]_i).     

Hemisynthesis,Antitumoral Effect,and Molecular Docking Studies of Ferutinin and Its Analogues

The natural product ferutinin was shown to act as an agonist to estrogen receptor ERα and agonist/antagonist to ERβ featuring a weak antiproliferative activity toward breast cancer cells. To enhance this activity, ferutinin analogues were synthesized by esterification of jaeschkenadiol with different acids. These compounds were assayed for their in vitro antiproliferative activity against estrogen‐dependent (MCF‐7) and estrogen‐independent (MDA‐MB‐231) breast cancer cell lines. Among the compounds, 3c’ exhibited a potent inhibitory selective activity against MCF‐7 with IC₅₀ value of 1 μm . Docking simulation of 3c’ in the ligand binding domain of the ERs indicated a potential antagonism interaction with both ER subtypes. Functional assay showed that 3c’ binds as an antagonist to ERα protein while ferutinin acts as an agonist.     

Stimulation of 5‐HT4 receptor enhances differentiation of mouse induced pluripotent stem cells into neural progenitor cells

Activation of serotonin (5‐hydroxytryptamine; 5‐HT) receptors plays a role in adult neurogenesis and differentiation of neural progenitor cells (NPC). Herein, we examined the involvement of 5‐HT receptors in the differentiation of mouse induced pluripotent stem (iPS) cells into NPC. To induce embryoid body (EB) formation, mouse iPS cells were cultured on ultralow‐attachment dishes. All‐trans retinoic acid (ATRA; 1 μmol/L) and/or 5‐HT (0.03 or 0.1 μmol/L) was added to the EB cultures for 4 days and then EB plated on gelatin‐coated plates were cultured for 7 or 14 days. Immunofluorescence staining revealed that mouse iPS cells expressed both 5‐HT_2A and 5‐HT₄ receptors and, to a lesser extent, 5‐HT_1A receptors. Treatment with 5‐HT significantly enhanced the ATRA‐induced expression of nestin, a specific marker for NPC, and phosphorylation of cAMP response element‐binding protein (CREB). Pretreatment of EB cultures with either 1 μmol/L GR113808 (a selective 5‐HT₄ receptor antagonist) or 1 μmol/L H89 (a protein kinase (PKA) inhibitor) significantly inhibited these effects of 5‐HT. These findings suggest that stimulation of 5‐HT₄ receptors may enhance ATRA‐induced neural differentiation of mouse iPS cells through activation of PKA and CREB.     

Vasorelaxation induced by methyl cinnamate,the major constituent of the essential oil of Ocimum micranthum,in rat isolated aorta

The aim of the present study was to investigate the vascular effects of the E‐isomer of methyl cinnamate (E‐MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1–3000 μmol/L, E‐MC concentration‐dependently relaxed endothelium‐intact aortic preparations that had been precontracted with phenylephrine (PHE; 1 μmol/L), with an IC₅₀ value (geometric mean) of 877.6 μmol/L (95% confidence interval (CI) 784.1–982.2 μmol/L). These vasorelaxant effects of E‐MC remained unchanged after removal of the vascular endothelium (IC₅₀ 725.5 μmol/L; 95% CI 546.4–963.6 μmol/L) and pretreatment with 100 μmol/L N^G‐nitro‐l ‐arginine methyl ester (IC₅₀ 749.0 μmol/L; 95% CI 557.8–1005.7 μmol/L) or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (IC₅₀ 837.2 μmol/L; 95% CI 511.4–1370.5 μmol/L). Over the concentration range 1–3000 μmol/L, E‐MC relaxed K⁺‐induced contractions in mesenteric artery preparations (IC₅₀ 314.5 μmol/L; 95% CI 141.9–697.0 μmol/L) with greater potency than in aortic preparations (IC₅₀ 1144.7 μmol/L; 95% CI 823.2–1591.9 μmol/L). In the presence of a saturating contractile concentration of K⁺ (150 mmol/L) in Ca²⁺‐containing medium combined with 3 μmol/L PHE, 1000 μmol/L E‐MC only partially reversed the contractile response. In contrast, under similar conditions, E‐MC nearly fully relaxed PHE‐induced contractions in aortic rings in a Ba²⁺‐containing medium. In preparations that were maintained under Ca²⁺‐free conditions, 600 and 1000 μmol/L E‐MC significantly reduced the contractions induced by exogenous Ca²⁺ or Ba²⁺ in KCl‐precontracted preparations, but not in PHE‐precontracted preparations (in the presence of 1 μmol/L verapamil). In addition, E‐MC (1–3000 μmol/L) concentration‐dependently relaxed the contractions induced by 2 mmol/L sodium orthovanadate. Based on these observations, E‐MC‐induced endothelium‐independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca²⁺ influx through voltage‐dependent Ca²⁺ channels. However, the involvement of a myogenic mechanism in the effects of E‐MC is also possible.     

Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation

1. The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca²⁺]_i) was studied in rat aorta. Cyclic AMP-elevating agents used were a β-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram).
2. In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca²⁺]_i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved.
3. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca²⁺]_i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta.
4. The combination of 5 μM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H-89 and not significantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell.
5. These findings support a role for both PKA and PKG in cyclic AMP-mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

     

Effects of ANP on pulmonary vein electrophysiology,Ca2+ homeostasis and adrenergic arrhythmogenesis via PKA

Atrial fibrillation (AF) is the most common form of arrhythmia and increases the risk of stroke and heart failure (HF). Pulmonary veins (PVs) are important sources of triggers that generate AF, and calcium (Ca²⁺) overload participates in PV arrhythmogenesis. Neurohormonal activation is an important cause of AF. Higher atrial natriuretic peptide (ANP) level predicts paroxysmal AF occurrence in HF patients. However, it is not clear if ANP directly modulates electrophysiological characteristics and Ca²⁺ homeostasis in the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the Fluo-3 fluorimetric ratio technique were performed using isolated rabbit PV preparations or single isolated PV cardiomyocytes before and after ANP administration. We found that ANP (1, 10, and 100 nmol/L) concentration-dependently decreased spontaneous activity in PV preparations. ANP (100 nmol/L) decreased isoproterenol (1 μmol/L)-induced PV spontaneous activity and burst firing. AP811 (100 nmol/L, NPR-C agonist), H89 (1μmol/L, PKA inhibitor) decreased isoproterenol-induced PV spontaneous activity or burst firing, but successive administration of ANP had no further effect on PV activity. KT5823 (1 μmol/L, PKG inhibitor) decreased isoproterenol-induced PV spontaneous activity but did not change isoproterenol-induced PV burst firing, whereas successive administration of ANP did not change isoproterenol-induced PV burst firing. ANP decreased intracellular Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content in single PV cardiomyocytes. ANP decreased the late sodium current, L-type Ca²⁺ current, but did not change nickel-sensitive Na⁺-Ca²⁺ exchanger current in single PV cardiomyocytes. In conclusion, ANP directly regulates PV electrophysiological characteristics and Ca²⁺ homeostasis and attenuates isoproterenol-induced arrhythmogenesis through NPR-C/cAMP/PKA signal pathway.     

Depotentiation of intact rat cardiac muscle unmasks an Epac‐dependent increase in myofilament Ca2+ sensitivity

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3′,5′‐monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation–contraction coupling in ventricular preparations from rat hearts. The study used 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′, 5′‐cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca²⁺ spark frequency from about 7 to 32/100 μm³/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca²⁺ and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca²⁺]_o) revealed no effect of Epac activation on either the amplitude of Ca²⁺ transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura‐2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm² vs cpTOME 23 ± 5 mN/mm², P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca²⁺]_o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca²⁺ sensitivity, an effect that was blocked by addition of KN‐93, a Ca²⁺/calmodulin‐dependent protein kinase II (CaMK‐II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.     

Effects of nitric oxide on large‐conductance Ca2+‐activated K+ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

The human cardiac fibroblast (HCF) is the most abundant cell type in the myocardium, and HCFs play critical roles in maintaining normal cardiac function. However, unlike cardiomyocytes, the electrophysiology of HCFs is not well established. In the cardiovascular system, Ca²⁺‐activated K⁺ (KCa) channels have distinct physiological and pathological functions, and nitric oxide (NO) plays a key role. In this study, we investigated the potential effects of NO on KCa channels in HCFs. We recorded strong oscillating, well‐maintained outward K⁺ currents without marked inactivation throughout the test pulse period and detected outward rectification in the I‐V curve; these are all characteristics that are typical of KCa currents. These currents were blocked with iberiotoxin (IBTX, a BKCa blocker) but not with TRAM‐34 (an IKCa blocker). The amplitudes of the currents were increased with SNAP (an NO donor), and these increases were inhibited with IBTX. The SNAP‐stimulating effect on the BKCa currents was blocked by pretreatment with KT5823 (a protein kinase G [PKG] inhibitor) or 1 H‐[1,‐2, ‐4] oxadiazolo‐[4,‐3‐a] quinoxalin‐1‐one (ODQ; a soluble guanylate cyclase inhibitor). Additionally, 8‐bromo‐cyclic guanosine 3’,5’‐monophosphate (8‐Br‐cGMP) stimulated the BKCa currents, and pretreatment with KT5720 (a protein kinase A [PKA] inhibitor) and SQ22536 (an adenylyl cyclase inhibitor) blocked the NO‐stimulating effect on the BKCa currents. Furthermore, 8‐bromo‐cyclic adenosine 3’,5’‐monophosphate (8‐Br‐cAMP) activated the BKCa currents. These data suggest that BKCa current is the main subtype of the KCa current in HCFs and that NO enhances these currents through the PKG and PKA pathways.     

Signalling pathways involved in p47phox‐dependent reactive oxygen species in platelets of endotoxemic rats

Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species (ROS) by different cell types contributes to sepsis. Platelets generate ROS, but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide (LPS)‐treated rats to investigate the p47^phox activation and ROS generation, and its modulation by c‐Src family kinase (c‐Src), phosphoinositide 3‐kinase (PI3K), protein kinase C (PKC) and protein kinase G (PKG). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre‐incubated with different inhibitors and subsequently activated or not with ADP. Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47^phox phosphorylation and ROS generation compared with the control group (P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non‐selective SFKs inhibitor PP2, the PI3K inhibitor wortmannin or the Akt inhibitor PPI‐1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group (P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ, the PKG inhibitor Rp‐8‐Br and the PKC inhibitor GF109203X abrogated the increased p47^phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP‐PKG and PKC‐p47^phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.     

CCN2 and CCN5 exerts opposing effect on fibroblast proliferation and transdifferentiation induced by TGF‐β

Epidural fibrosis might occur after lumbar discectomy and contributes to failed back syndrome. Transforming growth factor (TGF)‐β has been reported to influence multiple organ fibrosis, in which connective tissue growth factor/cysteine‐rich 61/nephroblastoma overexpressed 2 (CCN2) and CCN5 are involved. However, the effect of CCN2 and CCN5 on TGF‐β induced fibrosis has not yet been elucidated. This study reports that CCN2 and CCN5 play opposing roles in cell proliferation and transdifferentiation of human skin fibroblasts or rabbit epidural scar‐derived fibroblasts exposed to TGF‐β. We observed that TGF‐β1 induced fibroblasts proliferation and differentiation in a dose‐dependent manner (from 0 μg/L to 20 μg/L). Meanwhile, CCN2 expression is up‐regulated while CCN5 expression is inhibited by TGF‐β1 exposure. Furthermore, it is demonstrated that CCN2 overexpression leads to promoted proliferation and elevated collagen and α‐smooth muscle actin (α‐SMA) expression, which are inhibited by CCN5 overexpression. Moreover, it is shown that the cysteine knot (CT) domain, present in CCN2 but absent in CCN5, plays an essential part in fibroblast proliferation and differentiation. Additionally, enhanced TGF‐β and CCN2 expression but decreased CCN5 expression is found in rabbit epidural scar‐derived fibroblasts. Overall, the results show the opposing effects of CCN2 and CCN5 on fibroblast proliferation and transdifferentiation induced by TGF‐β.     

Effects of long‐term application of metoprolol and propranolol in a rat model of smoking

Beta‐blockers, especially selective β₁‐adrenoceptor antagonists, are often used to treat cardiovascular disease, even when complicated by chronic obstructive pulmonary disease. The association of beta‐blocker selectivity and treatment effects is disputed, and the curative effects and side‐effects of various antagonists may differ. Herein we investigated the effects of 1 months treatment with the selective β₁‐adrenoceptor antagonist metoprolol and the non‐selective β‐adrenoceptor antagonist propranolol on pulmonary function and pathology in a 4‐month rat model of passive cigarette smoke exposure and explored potential mechanisms of action. Lung function and general pathological changes were evaluated after 4 months exposure to cigarette smoke, with metoprolol and propranolol treatment (50 and 25 mg/kg per day, respectively; intragastrically) during the last month. Cytokine and mucin levels in bronchoalveolar lavage fluid (BALF) were determined by ELISA, whereas β₁‐ and β₂‐adrenoceptor expression in the lungs was evaluated by immunohistochemistry and western blot analysis. Chronic treatment with metoprolol and propranolol did not exacerbate peak expiratory flow or intra‐airway pressure in rats exposed to cigarette smoke. Propranolol significantly attenuated inflammatory cell infiltration, cytokine levels (tumour necrosis factor‐α and interleukin‐8) in BALF or mucus secretion, whereas metoprolol reduced only smooth muscle proliferation. Moreover, propranolol treatment was associated (albeit not significantly) with restoring β₂‐adrenoceptor expression in airway epithelia. Propranolol had a more beneficial effect on cigarette smoking‐induced lung damage than metoprolol in a smoking rat model that may be associated with restoration of endogenous β₂‐adrenoceptor density in the airway epithelial cells.     

Association of inflammation,myocardial fibrosis and cardiac remodelling in patients with mild aortic stenosis as assessed by biomarkers and echocardiography

相似文献   

Carbon monoxide activates large-conductance calcium-activated potassium channels of human cardiac fibroblasts through various mechanisms

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca²⁺-activated K⁺ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-N^G-monomethyl arginine citrate and L-N^G-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.     

B‐Type natriuretic peptide suppression of neutrophil superoxide generation: mechanistic studies in normal subjects

Many acute cardiovascular disease states are associated with neutrophil infiltration of myocardium and subsequent release of superoxide (O₂^?) and myeloperoxidase (MPO), which contribute to inflammatory reactions. B‐Type natriuretic peptide (BNP) is known to exert anti‐inflammatory and antifibrotic effects, but it is not known whether these may include interactions with neutrophils. In neutrophils isolated from 20 healthy subjects, we assessed the effect of BNP on the ‘neutrophil burst’ (O₂^? production and MPO release) stimulated by phorbol myristate acetate (PMA) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), respectively. Effects of BNP on cGMP accumulation, and the effects of the cell‐permeable cGMP analogue 8‐(4‐chlorophenylthio) guanosine‐cGMP (8‐p‐CPT‐cGMP) and protein kinase G (PKG) inhibition with KT5823 on the neutrophil–BNP interaction were also evaluated. B‐Type natriuretic peptide suppressed O₂^? release from neutrophils by 23 ± 6% (P < 0.001) and 24 ± 8% (P < 0.05) following PMA and fMLP stimulation, respectively. Although BNP did not significantly increase cGMP formation, 8‐p‐CPT‐cGMP suppressed both PMA‐ and fMLP‐induced neutrophil O₂^? release by 16% and 28%, respectively (P < 0.05). The PKG inhibitor KT5823 attenuated the effects of BNP on both fMLP‐ and PMA‐associated O₂^? production. Neither BNP nor 8‐p‐CPT‐cGMP significantly affected MPO release from neutrophils. Suppression of O₂^? release from neutrophils by BNP may contribute to its anti‐inflammatory and antifibrotic actions.     

Methadone is a Non‐Competitive Antagonist at the α4β2 and α3* Nicotinic Acetylcholine Receptors and an Agonist at the α7 Nicotinic Acetylcholine Receptor

Nicotine–methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non‐competitive antagonist at rat α3β4 nicotinic acetylcholine receptors (nAChR) and an agonist at human α7 nAChRs. In this study, we used cell lines expressing human α4β2, α7 and α3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [³H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and ⁸⁶Rb⁺ efflux and changes in intracellular calcium [Ca²⁺]_i were used to assess changes in the functional activity of the receptors. Methadone displaced [³H]epibatidine from nicotinic agonist‐binding sites in SH‐EP1‐hα7 and SH‐SY5Y cells, but not in SH‐EP1‐hα4β2 cells. The K_i values for methadone were 6.3 μM in SH‐EP1‐hα7 cells and 19.4 μM and 1008 μM in SH‐SY5Y cells. Methadone increased [Ca²⁺]_i in all cell lines in a concentration‐dependent manner, and in SH‐EP1‐hα7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH‐EP1‐hα4β2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre‐treatment abolished the nicotine‐induced response in [Ca²⁺]_i in all cell lines expressing nAChRs. In SH‐EP1‐hα4β2 and SH‐SY5Y cells, methadone had no effect on the ⁸⁶Rb⁺ efflux, but it antagonized the nicotine‐induced ⁸⁶Rb⁺ ion efflux in a non‐competitive manner. These results suggest that methadone is an agonist at human α7 nAChRs and a non‐competitive antagonist at human α4β2 and α3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co‐administration in human beings and might be of interest to the field of drug discovery.     

Expression in Escherichia coli of fusion protein comprising α‐conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

Nicotinic acetylcholine receptors (nAChRs) are ligand‐gated ion channels, which are widely distributed in the central and peripheral nervous system. The α6β2* nAChR is an important subtype, which is closely associated with nicotine addiction and movement disorders etc. α‐conotoxin TxIB with 16‐amino acid residues specifically targets α6β2* nAChR with no obvious effect on other nAChR subtypes. However, chemical synthesis of TxIB is expensive, and the quantity of native TxIB extracted from cone snail is limited. In the present study, we attempted to obtain TxIB using biological method based on the recombinant expression in Escherichia coli (E. coli). The synthetic gene encoding mature peptide of TxIB was inserted in pET‐31b(+) vector and transformed into E. coli strain BLR(DE3)pLysS for expression. The recombinant fusion protein KSI‐TxIB‐His₆ (KSI, ketosteroid isomerase) was expressed successfully as inclusion body in E. coli, which was purified by Ni‐NTA affinity chromatography column and cleaved by cyanogen bromide (CNBr) to release recombinant α‐conotoxin TxIB (rTxIB). Then, rTxIB was purified by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) and was identified by electrospray ionization mass spectrometry (ESI‐MS). Pharmacological activity of rTxIB was assessed by electrophysiological approaches. The results indicated that it preserved about 50% of potency, but, was even more important, had the same selectivity as the natural conotoxin which may provide an alternative method for quantity production of small peptides with low cost on the premise of not changing their potency.     

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Background and Purpose

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca²⁺ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity.

Experimental Approach

To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells.

Key Results

We showed that istaroxime enhances SERCA2a activity, Ca²⁺ uptake and the Ca²⁺-dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca²⁺ cycling.

Conclusions and Implications

Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca²⁺ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.