Deprogramming metabolism in pancreatic cancer with a bi-functional GPR55 inhibitor and biased ß2 adrenergic agonist.

Metabolic reprogramming contributes to oncogenesis, tumor growth, and treatment resistance in pancreatic ductal adenocarcinoma (PDAC). Here we report the effects of (R,S')-4'-methoxy-1-naphthylfenoterol (MNF), a GPR55 antagonist and biased ß2-adrenergic receptor (ß2-AR) agonist on cellular signaling implicated in proliferation and metabolism in PDAC cells. The relative contribution of GPR55 and ß2-AR in (R,S')-MNF signaling was explored further in PANC-1 cells. Moreover, the effect of (R,S')-MNF on tumor growth was determined in a PANC-1 mouse xenograft model. PANC-1 cells treated with (R,S')-MNF showed marked attenuation in GPR55 signal transduction and function combined with increased ß2-AR/Gαs/adenylyl cyclase/PKA signaling, both of which contributing to lower MEK/ERK, PI3K/AKT and YAP/TAZ signaling. (R,S')-MNF administration significantly reduced PANC-1 tumor growth and circulating L-lactate concentrations. Global metabolic profiling of (R,S')-MNF-treated tumor tissues revealed decreased glycolytic metabolism, with a shift towards normoxic processes, attenuated glutamate metabolism, and increased levels of ophthalmic acid and its precursor, 2-aminobutyric acid, indicative of elevated oxidative stress. Transcriptomics and immunoblot analyses indicated the downregulation of gene and protein expression of HIF-1α and c-Myc, key initiators of metabolic reprogramming in PDAC. (R,S')-MNF treatment decreased HIF-1α and c-Myc expression, attenuated glycolysis, shifted fatty acid metabolism towards ß-oxidation, and suppressed de novo pyrimidine biosynthesis in PANC-1 tumors. The results indicate a potential benefit of combined GPR55 antagonism and biased ß2-AR agonism in PDAC therapy associated with the deprogramming of altered cellular metabolism.

Effect of dual tocolysis with fenoterol and atosiban in human myometrium.

Objectives To measure the tocolytic effect of the combination of the oxytocin receptor antagonist atosiban with the ß-mimetic agent fenoterol on human myometrium of pregnant women. Methods An in vitro study of contractility in human myometrium at the Laboratory of the Department of Obstetrics, University Hospital of Zürich, Switzerland, was performed. Thirty-six human myometrial biopsies were obtained during elective caesarean sections of singleton pregnancies at term. Tissue samples were exposed to atosiban, fenoterol and the combination of atosiban with fenoterol. Contractility was measured as area under the curve during 30 min of spontaneous contractions. The effect of treatment was expressed as the percentage of change from basal activity during 30 min of exposure. Differences were calculated using a paired Wilcoxon signed-rank test. An additive effect of dual tocolysis was assumed when no significant difference was detected between the observed and expected inhibition of dual tocolysis. When inhibition was greater or lower than expected, the dual combination was characterised as "synergistic" or "antagonistic", respectively. Results Atosiban and fenoterol alone suppressed contractions by a median of 43.2% and 29.8%, respectively. The combination of atosiban plus fenoterol was measured at a level of 67.3% inhibition. There was no significant difference in the expected (63.2%) and observed inhibition effect of dual tocolysis (P=0.945). Conclusion This study demonstrated an additive effect of dual tocolysis of atosiban and fenoterol on human myometrium in vitro, but no synergistic or antagonistic effect.

GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts.

The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-ß-catenin, and PI3K-AKT signaling pathways. (R,R')-4'-methoxy-1-naphthylfenoterol ((R,R')-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R')-MNF's effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R')-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of ß-hydroxybutyrate, carnitine, and l-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R')-MNF. Metabolomics data indicate that (R,R')-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of ß-hydroxybutyrate and carnitine while reducing l-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R')-MNF. Mice treated with (R,R')-MNF had significant accumulation of l-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective l-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, ß-catenin, hexokinase 2, and p-glycoprotein were also observed. The data suggest that (R,R')-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

Concurrent activation of ß2-adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells.

Activation of ß2-adrenergic receptor (ß2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a bivalent compound that agonizes ß2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent ß2AR activation and GPR55 blockade in C6 glioma cells using (R,R')-MNF as a marker ligand. Our data show that (R,R')-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R')-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of ß2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R')-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R')-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of ß-catenin occurred with (R,R')-MNF, and we provided new evidence of (R,R')-MNF-mediated inhibition of oncogenic ß-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of ß2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.

Anti-inflammatory activities of fenoterol through ß-arrestin-2 and inhibition of AMPK and NF-κB activation in AICAR-induced THP-1 cells.

The AMP-activated protein kinase (AMPK) pathway has been shown to be able to regulate inflammation in several cell lines. We reported that fenoterol, a ß2-adrenergic receptor (ß2-AR) agonist, inhibited lipopolysaccharide (LPS)-induced AMPK activation and inflammatory cytokine production in THP-1 cells, a monocytic cell line in previous studies. 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an agonist of AMPK. Whether AICAR induced AMPK activation and inflammatory cytokine production in THP-1 cells can be inhibited by fenoterol is unknown. In this study, we explored the mechanism of ß2-AR stimulation with fenoterol in AICAR-induced inflammatory cytokine secretion in THP-1 cells. We studied AMPK activation using p-AMPK and AMPK antibodies, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion in THP-1 cells stimulated by ß2-AR in the presence or absence of AICAR and small interfering RNA (siRNA)-mediated knockdown of ß-arrestin-2 or AMPKα1 subunit. AICAR-induced AMPK activation, NF-κB activation and tumor necrosis factor (TNF)-α release were reduced by fenoterol. In addition, siRNA-mediated knockdown of ß-arrestin-2 abolished fenoterol's inhibition of AICAR-induced AMPK activation and TNF-α release, thus ß-arrestin-2 mediated the anti-inflammatory effects of fenoterol in AICAR-treated THP-1 cells. Furthermore, siRNA-mediated knockdown of AMPKα1 significantly attenuated AICAR-induced NF-κB activation and TNF-α release, so AMPKα1 was a key signaling molecule involved in AICAR-induced inflammatory cytokine production. These data suggested that fenoterol inhibited AICAR-induced AMPK activation and TNF-α release through ß-arrestin-2 in THP-1 cells. Management especially inhibition of AMPK signaling may provide new approaches and strategies for the treatments of immune diseases including inflammatory diseases and other critical illness.

Selective GPR55 antagonism reduces chemoresistance in cancer cells.

G protein-coupled receptor 55 (GPR55) possesses pro-oncogenic activity and its function can be competitively inhibited with (R,R')-4'-methoxy-1-naphthylfenoterol (MNF) through poorly defined signaling pathways. Here, the anti-tumorigenic effect of MNF was investigated in the human pancreatic cancer cell line, PANC-1, by focusing on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) exporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Incubation of PANC1 cells with MNF (1µM) for 24h significantly decreased EGF receptor, pyruvate kinase M2 (PKM2), and ß-catenin protein levels and was accompanied by significant reduction in nuclear accumulation of HIF-1α and the phospho-active forms of PKM2 and ß-catenin. Inhibition of GPR55 with either MNF or the GPR55 antagonist CID 16020046 lowered the amount of MDR proteins in total cellular extracts while diminishing the nuclear expression of Pgp and BCRP. There was significant nuclear accumulation of doxorubicin in PANC-1 cells treated with MNF and the pre-incubation with MNF increased the cytotoxicity of doxorubicin and gemcitabine in these cells. Potentiation of doxorubicin cytotoxicity by MNF was also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells, which express high levels of GPR55. The data suggest that inhibition of GPR55 activity produces antitumor effects via attenuation of the MEK/ERK and PI3K-AKT pathways leading to a reduction in the expression and function of MDR proteins.

Activation of ß2-adrenergic receptor by (R,R')-4'-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells.

(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective ß2 adrenergic receptor (ß2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the ß2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the ß2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in ß2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon ß2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified ß2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in ß2-AR-expressing lesions.

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the ß2-adrenoceptor.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the ß2-adrenoceptor (ß2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using ß2AR-Gsα and ß2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via ß-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of ß2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased ß2AR agonists.

Wnt/ß-catenin signaling modulates human airway sensitization induced by ß2-adrenoceptor stimulation.

BACKGROUND: Regular use of ß2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to ß2-adrenoceptor agonists in human isolated airways. METHODS: Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a ß2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells. RESULTS: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/ß-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade. CONCLUSIONS: Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/ß-catenin pathway, suggesting a phenomenon of biased agonism in connection with the ß2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

BACKGROUND AND PURPOSE: Despite the view that only ß2- as opposed to ß1-adrenoceptors (ßARs) couple to G(i), some data indicate that the ß1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. EXPERIMENTAL APPROACH: We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ß2ARs (ß1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. KEY RESULTS: PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. CONCLUSIONS AND IMPLICATIONS: Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Increased response to ß2-adrenoreceptor stimulation augments inhibition of IKr in heart failure ventricular myocytes.

BACKGROUND: Increasing evidence indicates that the rapid component of delayed rectifier potassium current (I(Kr)) is modulated by α- and ß-adrenergic stimulation. However, the role and mechanism regulating I(Kr) through ß(2)-adrenoreceptor (ß-AR) stimulation in heart failure (HF) are unclear. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we investigated the effects of fenoterol, a highly selective ß(2)-AR agonist, on I(Kr) in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. I(Kr) was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in I(Kr). In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in I(Kr). These effects were not modified by the incubation of myocytes with CGP-20712A, a ß(1)-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a ß(2)-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of I(Kr) in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD(90)) repolarization in HF ventricular myocytes. CONCLUSIONS: The results indicate that inhibition of I(Kr) induced by ß(2)-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes.

Cannabinoid receptor activation correlates with the proapoptotic action of the ß2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.

Contribution of the extracellular cAMP-adenosine pathway to dual coupling of ß2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle.

ß(2)-Adrenoceptor (ß(2)-AR) agonists increase skeletal muscle contractile force via activation of G(s) protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of ß(2)-AR to G(s) and G(i) proteins and the influence of the ß(2)-AR/G(s)-G(i)/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the ß(2)-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (ß(2)-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10-1000 µM), 1 µM forskolin, and 20 µM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the ß(2)-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (G(i) signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of ß(2)-AR to G(i) protein which depends on cAMP efflux. Remarkably, the PTX-sensitive ß(2)-AR inotropic effect was inhibited by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5'-phosphodiesterase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt, indicating that ß(2)-AR coupling to G(i) is indirect and dependent on A(1) receptor activation. The involvement of the extracellular cAMP-adenosine pathway in ß(2)-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.

Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in chronic obstructive pulmonary disease.

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the ß(2)-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1.

Effect of fenoterol stereochemistry on the ß2 adrenergic receptor system: ligand-directed chiral recognition.

The ß(2) adrenergic receptor (ß(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a ß(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of ß(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts.

Inhibitory effects of albuterol and fenoterol on RANTES and IP-10 expression in bronchial epithelial cells.

Short-acting ß2-adrenoreceptor agonist (SABA) is the major asthma reliever as indicated in the GINA guidelines. Regulated on activation, normal T expressed and secreted (RANTES) is a chemokine that attracts eosinophils, mast cells, and basophils toward site of allergic inflammation. Interferon γ-inducible protein (IP)-10 is a Th1-related chemokine that is also important in asthmatic inflammation and also involved in our immune defense against pathogens. Bronchial epithelial cells are first-line barrier against invasive pathogen and also have immunomodulatory function. However, whether albuterol and fenoterol (two SABAs) have modulatory effects on RANTES and IP-10 expression in bronchial epithelial cells is unknown. The human bronchial epithelial cell lines, BEAS-2B cells, were pre-treated with different concentrations of albuterol, fenoterol or dibutyryl-cAMP (a cyclic AMP analog) before polyinosinic-polycytidylic acid (poly I:C) stimulation. In some condition, BEAS-2B cells were pre-treated with ICI-118551, a selective ß2-adrenoreceptor antagonist, 30 min before albuterol or fenoterol treatment. The levels of RANTES and IP-10 were measured by ELISA. Intracellular signaling was investigated using cAMP assay, mitogen-activated protein kinase (MAPK) inhibitor, nuclear factor (NF)-κB inhibitor, and western blot. Albuterol and fenoterol suppressed poly I:C-induced RANTES and IP-10 expression of BEAS-2B cells. ICI-118551 could partly reverse the suppressive effects of albuterol and fenoterol on RANTES and IP-10 expression. Albuterol and fenoterol increased intracellular cAMP levels. Dibutyryl-cAMP conferred the similar effects of albuterol and fenoterol. Western blot revealed that albuterol suppressed p-ERK, p-JNK and pp38, and also their associated kinase expression. Albuterol had no effect on pp65 expression. Albuterol and fenoterol could suppress poly I:C-induced RANTES and IP-10 expression in human bronchial epithelial cells via at least partly the ß2-adrenoreceptor-cAMP and the MAPK pathways, implicating that albuterol and fenoterol could exert anti-inflammatory effect and benefit asthmatic patients by suppressing RANTES and IP-10 expression. However, these suppressive effects of albuterol and fenoterol may inhibit the defense against viral infection.

{Beta}2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells.

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses ß(2)-adrenergic receptors (ß(2)-ARs) that are coupled to G(s) activation and consequent cAMP production. Experiments were conducted to determine whether the ß(2)-AR agonist (R,R')-fenoterol and other ß(2)-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize ß(2)-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R')-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [(3)H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of ß(2)-AR as determined by receptor binding. (R,R')-fenoterol and other ß(2)-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [(3)H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [(3)H]thymidine incorporation. U118 cells have a reduced number of ß(2)-ARs and a concomitant reduction in the ability of ß(2)-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of ß(2)-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain ß(2)-ARs to a greater extent than whole brain, (R,R')-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine ß(2)-AR expression.

[Mechanism of the slow inotropic response of the mouse atrium mediated by the beta2-adrenoreceptor].

The abundant beta2-adrenoceptors (AR) expression was revealed in the mouse atrial cardiomyocytes, albeit its function is poorly understood. Recently we revealed the slow developing (for 20-40 min) positive inotropic effect in the mouse atrium which was induced by the specific agonist of the beta2-adrenoceptors (5 mkM fenoterol) and the task of this study involved investigation of the found effect. It was shown that stimulation of beta2-AR is enough for rapid triggering of up-regulation of two signalling pathways that have the opposite influence on the contraction force. On one hand, activation of the adenylate cyclase--protein kinase A cascade occurs leading to increasing of intracellular Ca2+ concentration and amplitude of contraction; on the other hand, activation of the NO-synthase and enhance of NO production occurs which prevents the potentiating of the contraction force. During the first 15-20 minutes, superposition of these activation effects was revealed, which prevented the contraction strength increasing. Then the positive inotropic effects occurred due to the decreasing of NO production. It was shown that L-type Ca-channels and ryanodine receptors were the key targets incorporated in the beta2 adrenoreceptors signalling puzzle.

beta2-Agonist modulates epithelial gene expression involved in the T- and B-cell chemotaxis and induces airway sensitization in human isolated bronchi.

Regular use of beta(2)-adrenoceptor agonists may enhance non-specific airway responsiveness and inflammation. In earlier experimental studies, we showed that prolonged in vitro fenoterol exposure induced airway sensitization via perturbed epithelial regulation of bronchoconstriction. The aim of the present work was to examine the involvement of inflammatory mediator genes and proinflammatory cells and to investigate the role of the bronchial epithelium in these untoward effects. Bronchial tissues were surgically removed from 17 ex-smokers. Bronchial rings and primary cultures of bronchial epithelial cells were incubated with 0.1microM fenoterol for 15h. Levels of mRNA-expression were analyzed using a real-time quantitative reverse transcription-polymerase chain reaction array. Bronchial rings were contracted with endothelin-1 and immune cell infiltration was assessed by immunohistochemistry. Compared to paired controls, fenoterol up-regulated the mRNAs of cytokines/proteins implicated in the recruitment of T and B cells or the activation and proliferation of bronchial epithelial cells (CCL20/MIP-3alpha, FOXA2, PPAR-gamma) in isolated bronchi and in cultured epithelial cells. Fenoterol exposure significantly enhanced CD8(+)-T and differentiated CD138(+)-B-cells infiltration into the bronchi, especially the subepithelial area. Increase in CD8 or CD138 labeling-intensity strongly correlated with rise in maximal contraction to endothelin-1 induced by fenoterol exposure. In summary, our results show that fenoterol modulates the T and B cells chemotaxis possibly via the epithelial chemokine secretion in isolated bronchi from ex-smokers. They also suggest that the infiltration of resident T and B cells into the subepithelial area is associated with an increase in airway responsiveness due to fenoterol exposure.

Stereochemistry of an agonist determines coupling preference of beta2-adrenoceptor to different G proteins in cardiomyocytes.

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(2)-adrenoceptor, selectively activates G(s) protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G(s) signaling, their S,R isomers were able to activate both G(s) and G(i) proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G(s),G(i2), and G(i3) proteins. The inefficient G(i) signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the beta(2)-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.