Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1.

Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the ß2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human-mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar ß2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure-function data of OCT1 is not directly transferrable between substrates or species.

Heterodimerization With 5-HT2BR Is Indispensable for ß2AR-Mediated Cardioprotection.

RATIONALE: The ß2-adrenoceptor (ß2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the ß2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac ß2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. OBJECTIVE: Here, we aim to investigate the potential cardioprotective effect of ß2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of ß2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). METHODS AND RESULTS: Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the ß2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another ß2-agonist zinterol markedly promoted heterodimerization of ß2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and ß2-ARs enhanced ß2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated ß2-AR-stimulated Gi signaling and cardioprotection. CONCLUSIONS: These data demonstrate that the ß2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of ß2-ARs and 5-HT2BRs.

Validation of Fenoterol to Study ß2-Adrenoceptor Function in the Rat Urinary Bladder.

Fenoterol is a ß2-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by ß2-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via ß3-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study ß2-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC50 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the ß2-AR antagonist ICI 118,551 and the ß3-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC50 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC50 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess ß2-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the ß-AR.

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Comparison efficacy of randomized nebulized magnesium sulfate and ipratropium bromide/fenoterol in children with moderate to severe asthma exacerbation.

BACKGROUND: In Thailand, nebulized ipratropium bromide/fenoterol, is commonly used in addition to salbutamol for severe asthma exacerbation. Recently, nebulized MgSO4 is indicated in GINA 2015 as an additive treatment for severe cases. However, there is limited data showed the efficacy of both drugs in childhood severe asthma. The purpose of this study to compare efficacy and safety of nebulized MgSO4 and ipratropium bromide/fenoterol in moderate to severe asthma attacks. METHODS: In this a prospective, double-blind, randomized, controlled trial study, we enrolled thirty-three children, age ranged from 2 to 15 years old, with PRAM score ≥ 4 (moderate to severe asthma exacerbation) despite 3 doses of nebulized salbutamol. Each patient was randomized to receive either three doses of nebulized MgSO4 or nebulized ipratropium bromide/fenoterol every 30 minutes. The PRAM score was measured at 0, 30, 60, 90, 120 and 240 minutes after the treatment. The adverse event and admission days were also evaluated. RESULTS: Sixteen patients received nebulized MgSO4 and seventeen received nebulized ipratropium bromide/fenoterol. Almost patients were classified as having moderate asthmatic attack. There were no statistically significant difference between the two study groups in almost baseline characteristic, PRAM score at 0, 30, 60, 90, 120, 240 minutes. The hospital length of stay was also similar between two groups (p = 0.83). There were no serious events in both groups. CONCLUSIONS: Our double blind, randomized, controlled pilot study demonstrated non-inferior outcomes including clinical benefit and safety of nebulized MgSO4 and nebulized ipratropium bromide/fenoterol among Thai children with acute moderate asthmatic.

Hemi-Babim and Fenoterol as Potential Inhibitors of MPro and Papain-like Protease against SARS-CoV-2: An In-Silico Study.

The coronaviruses belong to the Coronaviridae family, and one such member, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is causing significant destruction around the world in the form of a global pandemic. Although vaccines have been developed, their effectiveness and level of protection is still a major concern, even after emergency approval from the World Health Organisation (WHO). At the community level, no natural medicine is currently available as a cure. In this study, we screened the vast library from Drug Bank and identified Hemi-Babim and Fenoterol as agents that can work against SARS-CoV-2. Furthermore, we performed molecular dynamics (MD) simulation for both compounds with their respective proteins, providing evidence that the said drugs can work against the MPro and papain-like protease, which are the main drug targets. Inhibiting the action of these targets may lead to retaining the virus. Fenoterol is a beta-2 adrenergic agonist used for the symptomatic treatment of asthma as a bronchodilator and tocolytic. In this study, Hemi-Babim and Fenoterol showed good docking scores of -7.09 and -7.14, respectively, and performed well in molecular dynamics simulation studies. Re-purposing the above medications has huge potential, as their effects are already well-proven and under public utilisation for asthma-related problems. Hence, after the comprehensive pipeline of molecular docking, MMGBSA, and MD simulation studies, these drugs can be tested in-vivo for further human utilisation.

Antenatal exposure to fenoterol is not associated with the development of retinopathy of prematurity in infants born before 32 weeks of gestation.

PURPOSE: Despite safety concerns, ß2-sympathomimetics are still widely used as tocolytic agents. ß-Blockers in turn are used to treat vasculo-proliferative diseases of the newborn such as retinopathy of prematurity (ROP), which may lead to visual impairment and blindness. The scope of this study was to investigate whether antenatal exposure to the ß2-sympathomimetic fenoterol contributes to the development of ROP. METHODS: For this single-center retrospective case-control study of prospectively collected clinical data, all infants born before 32 weeks of gestation between 2001 and 2012 were included. The association of prenatal exposure to fenoterol and the development of ROP were analyzed by multivariate logistic regression. RESULTS: n = 1134 infants < 32 weeks of gestation were screened for eligibility, out of which n = 722 met the inclusion criteria. Exposure to fenoterol (n = 505) was not associated with a higher rate of ROP (OR 0.721, 95% CI 0.463-1.122). Further, duration of exposure (days) did not alter the incidence of ROP (OR 1.001, 95% CI 0.986-1.016). Frequency distribution of different ROP stages and the need for therapeutic intervention was also not affected by prenatal exposure to fenoterol. Risk factors for the development of ROP like low birth weight, low gestational age, prolonged respiratory support and multiple gestation were confirmed in our large study cohort. CONCLUSION: ß2-Sympathomimetic tocolysis does not increase the rate of ROP in premature infants born < 32 weeks of gestation. Our results render fenoterol a safe tocolytic agent regarding neonatal ROP development.

Effect of betamethasone, indomethacin and fenoterol on neonatal and maternal mononuclear cells stimulated with Escherichia coli.

Despite considerable progress in the field of perinatal care, infectious diseases, especially when caused by gram negative bacteria, remain a major reason for neonatal morbidity and mortality. Notably infants born prematurely and those with very low birth weight are at risk due to their immature and deficient immune system and their prolonged hospitalization which promotes nosocomial infections. In case of impending preterm birth, betamethasone is given to induce lung maturation and tocolytic agents like indomethacin or fenoterol are administered to suppress premature labor. The aim of this study was to analyze the effects of these drugs on the immune system of mothers and neonates. Therefore, mononuclear cells from cord blood and peripheral maternal blood were stimulated with Escherichia coli and incubated with betamethasone, indomethacin and fenoterol. Subsequently the effect of the treatment on cytokine production was determined. Betamethasone alone and in combination with tocolytic agents inhibited the production of pro- and anti-inflammatory cytokines. Not only does betamethasone dampen the immune response by reducing the production of cytokines, it also has a variety of other detrimental short- and long-term effects on the neonate. In conclusion we would recommend using biological markers to determine if premature labor actually leads to preterm birth and subsequently administer betamethasone only to mothers giving birth prematurely.

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.

MicroRNA-150 Modulates Ischemia-Induced Neovascularization in Atherosclerotic Conditions.

OBJECTIVE: Hypercholesterolemia is an atherosclerotic condition that is associated with impaired neovascularization in response to ischemia. This study sought to define the role of microRNAs in that pathophysiology. APPROACH AND RESULTS: Next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses identified miR-150 as a proangiogenic microRNA, which expression is significantly reduced in the ischemic muscles of hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice, and in human umbilical vein endothelial cells exposed to oxidized low-density lipoprotein. Forced expression of miR-150 using a miR mimic could rescue oxidized low-density lipoprotein-mediated impairment of endothelial cell migration and tubule formation in vitro. In a mouse model of hindlimb ischemia, intramuscular injection of miR-150 mimic restored blood flow recuperation, vascular densities in ischemic muscles, and functional mobility in ApoE-/- mice. Treatment of ApoE-/- mice with miR-150 also increased the number and the activities of proangiogenic cells. miR-150 targets SRC kinase signaling inhibitor 1, an important regulator of Src (proto-oncogene tyrosine-protein kinase Src) activity. Here we found that hypercholesterolemia and oxidized low-density lipoprotein exposure are associated with increased SRC kinase signaling inhibitor 1 expression and decreased Src activity. However, treatment with miR-150 mimic reduces SRC kinase signaling inhibitor 1 expression and restores Src and downstream endothelial nitric oxide synthase and Akt (protein kinase B) activities both in vitro and in vivo. We also demonstrate the interrelation between miR-150 and SRC kinase signaling inhibitor 1 and their importance for endothelial cell angiogenic activities. CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Drug exposure and the risk of multiple sclerosis: A systematic review.

BACKGROUND: Several environmental and lifestyle factors have been associated with multiple sclerosis (MS) risk, including some pharmacological treatments. We systematically reviewed the literature on prescription drug exposure and MS risk. METHODS: Six databases were searched for original observational studies reporting drug exposure and MS risk published before 2017. RESULTS: Thirteen articles fulfilled inclusion criteria. Exposure to neither amiloride nor valproic acid was associated with MS (adjusted hazard ratio (adj.HR = 1.34;95% CI:0.81-2.20; adj.HR = 1.30;95%CI:0.44-3.80, respectively). Four studies explored oral contraceptive exposure and reported no association with MS; while a single study found an increased risk (odds ratio [adj.OR] = 1.52;95%CI:1.21-1.91). While penicillin exposure was associated with reduced risk of developing MS (adj.OR = 0.5;95%CI:0.3-0.9), a later study observed an elevated risk for penicillin (adj.OR = 1.21;95%CI:1.10-1.27) and all antibiotics (adj.OR = 1.41;95%CI:1.29-1.53), which was potentially attributed to underlying infection. Anti-tumor necrosis factor-alpha (TNFα) was not associated with MS risk in persons with inflammatory bowel disease (standard morbidity ratio = 4.2;95%CI:0.1-23.0) and arthritis (standardized incidence ratio = 1.38;95%CI:0.69-2.77); however, men exposed to anti-TNFα who also had arthritis and individuals with ankylosing spondylitis were at an increased risk (standardized incidence ratios = 3.91;95%CI:1.47-10.42 and 3.48;95%CI:1.45-8.37, respectively). A reduced risk of MS was observed with exposure to the beta2-adrenergic agonist fenoterol (adj.OR = 0.58;95%CI:0.45-0.76), and the sedating histamine 1-receptor antagonists (adj.OR = 0.2;95%CI:0.1-0.8), but not the non-sedating equivalent (adj.OR = 0.8;95%CI:0.4-1.6). CONCLUSIONS: The suggestion that some drugs may prevent MS is intriguing and warrants further study. In addition, further pharmacovigilance is needed to assess the safety of anti-TNFα drugs in specific populations in the context of MS risk.

Acute tocolysis for uterine tachysystole or suspected fetal distress.

BACKGROUND: Uterine tachysystole (more than 5 contractions per 10 minutes in 2 consecutive intervals) is common during labour, particularly with use of labour-stimulating agents. Tachysystole may reduce fetal oxygenation by interrupting maternal blood flow to the placenta during contractions. Reducing uterine contractions may improve placental blood flow, improving fetal oxygenation. This review aimed to evaluate the use of tocolytics to reduce or stop uterine contractions for improvement of the condition of the fetus in utero. This new review supersedes an earlier Cochrane Review on the same topic. OBJECTIVES: To assess the effects of the use of acute tocolysis during labour for uterine tachysystole or suspected fetal distress, or both, on fetal, maternal and neonatal outcomes. SEARCH METHODS: We searched Cochrane Pregnancy and Childbirth's Trials Register, ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP) (2 February 2018), and reference lists of retrieved studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) evaluating acute tocolysis for uterine tachysystole, intrapartum fetal distress, or both. DATA COLLECTION AND ANALYSIS: We used standard methods expected by Cochrane. MAIN RESULTS: We included eight studies (734 women), conducted in hospital settings, predominantly in high-income countries (USA, Austria, Uruguay). Two trials were conducted in upper and lower middle-income countries (South Africa, Sri Lanka). The hospital facilities all had the capacity to perform caesarean section. Overall, the studies had a low risk of bias, except for methods to maintain blinding. All of the trials used a selective beta2 (ß2)-adrenergic agonist in one arm, however the drug used varied, as did the comparator. Limited information was available on maternal outcomes.Selective ß2-adrenergic agonist versus no tocolytic agent, whilst awaiting emergency deliveryThere were two stillbirths, both in the no tocolytic control group (risk ratio (RR) 0.23, 95% confidence interval (CI) 0.01 to 4.55; 2 studies, 57 women; low-quality evidence). One had gross hydrocephalus and the second occurred with vaginal delivery after waiting 55 minutes for caesarean section. The decision for caesarean section delivery was an inclusion criterion in both studies so we could not assess this as an outcome under this comparison. Abnormal fetal heart trace is probably lower with tocolytic treatment (RR 0.28, 95% CI 0.08 to 0.95; 2 studies, 43 women; moderate-quality evidence). The effects on the number of babies with Apgar score below seven were uncertain (low-quality evidence).Intravenous (IV) atosiban versus IV hexoprenaline (1 study, 26 women) One infant in the hexoprenaline group required > 24 hours in the neonatal intensive care unit (NICU) following a forceps delivery (RR 0.33, 95% CI 0.01 to 7.50; low-quality evidence). There were no fetal or neonatal mortalities and no Apgar scores below seven. There was one caesarean delivery in the IV hexoprenaline group (RR 0.33, 95% CI 0.01 to 7.50; low-quality evidence), and one case of abnormal fetal heart score in the atosiban group (RR 3.00, 95% CI 0.13 to 67.51; very low-quality evidence).IV fenoterol bromhydrate versus emergency delivery (1 study, 390 women) No data were reported for perinatal death, severe morbidity or fetal or neonatal mortality. IV fenoterol probably increases the risk of caesarean delivery (RR 1.12, 95% CI 1.04 to 1.22; moderate-quality evidence). Fenoterol may have little or no effect on the risk of Apgar scores below seven (RR 1.28, 95% CI 0.35 to 4.68; low-quality evidence).IV hexoprenaline versus no tocolytic agent, whilst awaiting emergency delivery (1 study, 37 women) No data were reported for perinatal death or severe morbidity. There were two fetal deaths in the no tocolytic control group (RR 0.23, 95% CI 0.01 to 4.55; low-quality evidence). The rate of caesarean delivery was not reported. There were two babies with Apgar scores below seven in the control group and none in the hexoprenaline group (RR 0.24, 95% CI 0.01 to 4.57; 35 women; low-quality evidence).Subcutaneous terbutaline versus IV magnesium sulphate (1 study, 46 women)No data were reported for perinatal death, severe morbidity or fetal or neonatal mortality. The decision for caesarean section was an inclusion criterion, so we could not assess this. The effects on abnormal fetal heart trace are uncertain (very low-quality evidence).Subcutaneous terbutaline with continuation of oxytocic infusion versus cessation of oxytocic infusion without tocolytic agent (1 study, 28 women) No data were reported for perinatal death, severe morbidity or fetal or neonatal mortality. There may be little or no difference in the rates of caesarean delivery in the subcutaneous terbutaline (8/15) and control groups (4/13) (RR 1.73, 95% CI 0.68 to 4.45; low-quality evidence). There were no cases of Apgar scores below seven or abnormal fetal heart trace.Subcutaneous terbutaline versus no tocolytic agent, whilst awaiting emergency delivery (1 study, 20 women) No data were reported for perinatal death or severe morbidity. There were no fetal or neonatal mortalities. The decision for caesarean section was an inclusion criterion, so we could not assess this. There were two babies with Apgar scores below seven in the control group and none in the terbutaline group (RR 0.17, 95% CI 0.01 to 3.08; low-quality evidence).IV terbutaline versus IV nitroglycerin (1 study, 110 women)No data were reported for perinatal death or severe morbidity or fetal or neonatal mortality. There may be little or no difference in the rates of caesarean delivery between the IV terbutaline (30/57) and control groups (29/53) (RR 0.96, 95% CI 0.68 to 1.36; low-quality evidence). There were no cases of Apgar scores below seven. AUTHORS' CONCLUSIONS: There is insufficient evidence to determine the effects of tocolytics for uterine tachysystole or suspected fetal distress during labour. The clinical significance for some of the improvements in measures of fetal well-being with tocolytics is unclear. The sample sizes were too small to detect effects on neonatal morbidity, mortality or serious adverse effects. The majority of studies are from high-income countries in facilities with access to caesarean section, which may limit the generalisability of the results to lower-resource settings, or settings where caesarean section is not available.Further well-designed and adequately powered RCTs are required to evaluate clinically relevant indicators of maternal and neonatal morbidity and mortality.

Tocolysis with the ß2-sympathomimetic fenoterol does not increase the occurrence of infantile hemangioma in preterm and term infants.

PURPOSE: ß2-sympathomimetics are used in obstetrics as tocolytic agents, despite a remarkable profile of side effects. Recently, the ß2-sympathomimetic tocolytic drug hexoprenaline was identified as an independent risk factor for the development of infantile hemangioma (IH) in preterm infants. The aim of this study was to evaluate whether this observed effect was applicable to other ß2-mimetic tocolytic agents like fenoterol. METHODS: Clinical prospectively collected data of all infants born between 2001 and 2012 and admitted to the neonatal intensive care unit (NICU) at Heidelberg University Hospital and respective maternal data were merged. For the current retrospective cohort study, cases (IH) were matched to controls (no IH) at a ratio of 1:4, adjusting for birth weight, gestational age, gender and multiple gestations. Prenatal exposure to fenoterol and perinatal outcome were analyzed in the total cohort and in subgroups. RESULTS: N = 5070 infants were admitted to our neonatal department, out of which n = 172 infants with IH were identified and compared to n = 596 matched controls. Exposure to fenoterol was not associated with a higher rate of IH in the total matched population (OR 0.926, 95% CI 0.619-1.384) or in a subgroup of neonates < 32 weeks of gestation or with a birth weight < 1500 g (OR 1.127, 95% CI 0.709-1.791). In the total matched population, prenatal exposure to glucocorticoids was associated with a reduced occurrence of IH (OR 0.566, 95% CI 0.332-0.964) and neonates with IH showed a prolonged total hospital stay compared to controls (69 vs. 57 days, p = 0.0033). Known risk factors for IH were confirmed by our large study cohort and included female gender, low birth weight, preterm birth and multiple gestations (all p < 0.005). CONCLUSIONS: Exposure to fenoterol during pregnancy does not increase the occurrence of IH. Further studies are needed to explore differences in the risk profiles of different ß2-sympathomimetic tocolytic drugs.

Cholesterol regulates contractility and inotropic response to ß2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein-Akt-NO-pathway.

Majority of cardiac ß2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-ß-cyclodextrin (MßCD, 1 and 5mM) interferes with contractility and inotropic effect of ß2-adrenergic agonist (fenoterol, 50µM) in the mouse atria. Treatment with MßCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MßCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MßCD, pertussis toxin or Akt inhibitor could recover the ß2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MßCD-induced changes in the ß2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to ß2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the ß2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

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.

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.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

Bone morphogenetic protein receptor type II deficiency and increased inflammatory cytokine production. A gateway to pulmonary arterial hypertension.

RATIONALE: Mutations in bone morphogenetic protein receptor type II (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (PAH). However, disease penetrance is only 20-30%, suggesting a requirement for additional triggers. Inflammation is emerging as a key disease-related factor in PAH, but to date there is no clear mechanism linking BMPR-II deficiency and inflammation. OBJECTIVES: To establish a direct link between BMPR-II deficiency, a consequentially heightened inflammatory response, and development of PAH. METHODS: We used pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations and compared them with wild-type controls. For the in vivo model, we used mice heterozygous for a null allele in Bmpr2 (Bmpr2(+/-)) and wild-type littermates. MEASUREMENTS AND MAIN RESULTS: Acute exposure to LPS increased lung and circulating IL-6 and KC (IL-8 analog) levels in Bmpr2(+/-) mice to a greater extent than in wild-type controls. Similarly, pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations produced higher levels of IL-6 and KC/IL-8 after lipopolysaccharide stimulation compared with controls. BMPR-II deficiency in mouse and human pulmonary artery smooth muscle cells was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase. Chronic lipopolysaccharide administration caused pulmonary hypertension in Bmpr2(+/-) mice but not in wild-type littermates. Coadministration of tempol, a superoxide dismutase mimetic, ameliorated the exaggerated inflammatory response and prevented development of PAH. CONCLUSIONS: This study demonstrates that BMPR-II deficiency promotes an exaggerated inflammatory response in vitro and in vivo, which can instigate development of pulmonary hypertension.

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through ß-arrestin-2 in THP-1 cell line.

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a ß2-adrenergic receptor (ß2-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of ß2-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and ß-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of ß-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release, thus ß-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1ß release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the ß2-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1ß release via ß-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases.

Agonist binding by the ß2-adrenergic receptor: an effect of receptor conformation on ligand association-dissociation characteristics.

The ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR), is a physiologically important transmembrane protein that is a target for drugs used for treatment of asthma and cardiovascular diseases. Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association-dissociation process to and from the different conformational forms of ß2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist ((R,R)-fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from ß2-AR. The two different conformational forms of ß2-AR, i.e. active ß2-AR-PDB: 3P0G and inactive ß2-AR-PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of ß2-AR. Comparison of the residues surrounding these transient binding sites in both ß2-AR states revealed the importance of the aromatic residues F194, H93(2.64), H296(6.58), and H178 (extracellular part of ß2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of ß2-AR, depending on the conformation of the receptor.