μM Propranolol Research Articles

Opposite Contractile Effects of Amphetamine-Related Hallucinogenic Drugs in the Isolated Human Atrium.

The present study examined three hallucinogenic amphetamine derivatives, namely, 2,5-dimethoxy-4-iodoamphetamine (DOI) as well as 2,5-dimethoxy-4-methylamphetamine (DOM) and 4-methylmethcathinone (mephedrone). The objective of this study was to test the hypothesis that DOI, DOM, and mephedrone would increase the contractile force in isolated human atrial preparations in a manner similar to amphetamine. To this end, we measured contractile force under isometric conditions in electrically stimulated (1 Hz) human atrial preparations obtained during open surgery. DOI and DOM alone or in the presence of isoprenaline reduced the contractile force concentration-dependently in human atrial preparations. These negative inotropic effects of DOM and DOI were not attenuated by 10 µM atropine. However, mephedrone increased the contractile force in human atrial preparations in a concentration- and time-dependent manner. Furthermore, these effects were attenuated by the subsequent addition of 10 µM propranolol or pretreatment with 10 µM cocaine in the organ bath. Therefore, it can be concluded that amphetamine derivatives may exert opposing effects on cardiac contractile force. The precise mechanism by which DOI and DOM exert their negative inotropic effects remains unknown at present. The cardiac effects of mephedrone are probably due to the release of cardiac noradrenaline.

Contractile effects of stimulation of D1-dopamine receptors in the isolated human atrium.

Dopamine receptors have been claimed not to directly increase contractility in the human heart. Therefore, we performed contraction experiments in isolated electrically driven human atrial preparations (HAP). For comparison, we performed contraction experiments with left atrial preparations of transgenic mice which harbor a cardiac overexpression of human D1-dopamine receptors (D1-TG). In D1-TG, first we noted that dopamine (10nM-10µM cumulatively applied) in the presence of propranolol exerted a concentration- and time-dependent positive inotropic effect in D1-TG. In a similar fashion, dopamine increased force of contraction in the presence of 0.4µM propranolol in HAP and these effects were amplified by pre-treatment with inhibitor of phosphodiesterase III (1µM) cilostamide. Moreover, contractile effects of dopamine in the presence of propranolol 0.4µM in HAP were antagonized by odapipam, haloperidol, or raclopride. Ten micromolars of fenoldopam in the presence of cilostamide increased force of contraction in HAP and this effect was antagonized by SCH23390. We conclude that stimulation of human D1-dopamine receptors can increase force of contraction in the HAP.

Initial characterization of a transgenic mouse with overexpression of the human D1-dopamine receptor in the heart

Dopamine can exert effects in the mammalian heart via five different dopamine receptors. There is controversy whether dopamine receptors increase contractility in the human heart. Therefore, we have generated mice that overexpress the human D1-dopamine receptor in the heart (D1-TG) and hypothesized that dopamine increases force of contraction and beating rate compared to wild-type mice (WT). In D1-TG hearts, we ascertained the presence of D1-dopamine receptors by autoradiography using [3H]SKF 38393. The mRNA for human D1-dopamine receptors was present in D1-TG hearts and absent in WT. We detected by in-situ-hybridization mRNA for D1-dopamine receptors in atrial and ventricular D1-TG cardiomyocytes compared to WT but also in human atrial preparations. We noted that in the presence of 10 µM propranolol (to antagonize β-adrenoceptors), dopamine alone and the D1- and D5-dopamine receptor agonist SKF 38393 (0.1–10 µM cumulatively applied) exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in left or right atrial preparations from D1-TG. The positive inotropic effects of SKF 38393 in left atrial preparations from D1-TG led to an increased rate of relaxation and accompanied by and probably caused by an augmented phosphorylation state of the inhibitory subunit of troponin. In the presence of 0.4 µM propranolol, 1 µM dopamine could increase left ventricular force of contraction in isolated perfused hearts from D1-TG. In this model, we have demonstrated a positive inotropic and chronotropic effect of dopamine. Thus, in principle, the human D1-dopamine receptor can couple to contractility in the mammalian heart.

Contractile Effects of Amphetamine, Pseudoephedrine, Nor-pseudoephedrine (Cathine), and Cathinone on Atrial Preparations of Mice and Humans.

Amphetamine derivatives are used worldwide legally or illegally and intoxications may be accompanied by cardiac arrhythmias. Here, we tested contractile effects of cumulative applied (±)-amphetamine, pseudoephedrine, nor-pseudoephedrine (cathine), and cathinone in electrically stimulated (1 Hz) human right atrial preparations (HAP) and mouse left atrial preparations and in spontaneously beating mouse right atrial preparations. In mouse atrial preparations, amphetamine increased force of contraction and beating rate in a concentration- and time-dependent manner, starting at 1 µM in left atrial preparations to 157.1% ± 3.0% and right atrial preparations to 146.6% ± 9.8% at 10 µM, respectively [mean ± standard error of the mean (SEM); n = 5; P < 0.05]. Pseudoephedrine, cathine, or cathinone alone were ineffective in mouse atrial preparations but after pre-incubation with the phosphodiesterase IV inhibitor rolipram (0.1 µM), a positive inotropic effect was noted (mean ± SEM: pseudoephedrine: 112.3% ± 9.8%; cathine: 109.0% ± 4.3%; cathinone: 138.3% ± 21.2%). The effects of all drugs were greatly attenuated by 10 µM cocaine or 10 µM propranolol treatments. However, In HAP, not only amphetamine (to a mean ± SEM of 208% ± 32%) but also pseudoephedrine (to a mean ± SEM of 287% ± 60%), cathine (to a mean ± SEM of 234% ± 52%), and cathinone (to a mean ± SEM of 217% ± 65%) increased force of contraction without the need of phosphodiesterase inhibition. The contractile effects in HAP were attenuated by 10 µM cocaine and antagonized by 10 µM propranolol. We conclude that amphetamine, pseudoephedrine, cathine, and cathinone act probably via release of noradrenaline from cardiac stores as indirect sympathomimetic agents in mouse and more pronounced in human atrial preparations.

Rapid Droplet Sampling Interface for Low-Volume, High-Throughput Mass Spectrometry Analysis.

Here, we present a rapid droplet sampling interface (RDSI) electrospray ionization mass spectrometry (ESI-MS) system as a high-throughput, low-volume, noncontact, and minimal-carryover approach for characterization of liquids. Liquid characterization was achieved by combining droplet ejection with an open-face microflow capillary with a 2.5 μL/min continuous flow of carrier solvent. Through this implementation, single 0.3 nL droplets containing the analyte effectively mix with 4-8 nL of carrier solvent and create a combined electrospray plume. The carrier solvent continuously cleaned the system, eliminating carryover. A sampling rate of 5 Hz was achieved for droplets containing 1 μM propranolol or 5 μM leu-enkephalin with each droplet fully baseline-resolved (138 ± 32 ms baseline-to-baseline). Using a SCIEX API4000 mass spectrometer, a lower limit of quantification (LLOQ) of propranolol was 15 nM, corresponding to 1.16 fg of propranolol in the droplet, and was linear across 3 orders of magnitude. Quantitation could be achieved by adding an isotopically labeled internal standard, as done in conventional ESI. Signal transients were faster than the acquisition speed of the mass spectrometer, resulting in artificially high reproducibility of 15-30% RSD droplet-to-droplet. Analyte-solvent mixing ratios could be controlled by adjusting droplet positioning along the open-face capillary with an optimal position about 0.4 mm from the tip end. The range of analyte coverage was exemplified by measures of peptides and drugs in methanol, water, and buffer solutions. In a comparison to the Open Port Sampling Interface (OPSI) implemented on the same system, the RDSI had 78× greater sensitivity, 6× greater throughput and used significantly less carrier solvent.

The effects of propranolol on the biology and Notch signaling pathway of human umbilical vein endothelial cells.

Propranolol is the first choice for treating infantile hemangioma (IH). How propranolol works in IH remains unclear. Infantile hemangioma endothelial cells (HemECs) express Notch1, Jagged, Hey1, and other molecules in the Notch pathway, suggesting that Notch pathway-related molecules play an important role in affecting vascular endothelial cell proliferation. Whether propranolol can affect the Notch signaling pathway in IH treatment is unclear. We performed this study to observe the effect of propranolol on the expression of Notch signaling pathway molecules in human umbilical vein endothelial cells (HUVECs) and to explore the therapeutic mechanism of propranolol on IH. HUVECs cultured in vitro were exposed to 60, 120, 240, 360, or 480 µM propranolol. The morphological changes of the HUVECs were observed under an inverted microscope. HUVECs proliferation was detected with Cell Counting Kit-8 (CCK-8). The effects of propranolol on HUVECs apoptosis were detected by flow cytometry. The role of Notch in propranolol inhibition of HUVEC proliferation was analyzed with real-time polymerase chain reaction (PCR) and western blotting. Propranolol reduced HUVECs numbers and altered their morphology. The inhibitory effect of propranolol on cell proliferation was dependent on the reaction time and drug concentration. Propranolol upregulated Jagged1, Notch1, and Hey1 expression and downregulated delta-like ligand4 (DLL4) expression. Propranolol may play a role in IH treatment by increasing Jagged1 expression in endothelial cells, activating the Notch pathway and inducing the upregulation of the downstream target gene HEY1.

Methamphetamine increases force of contraction in isolated human atrial preparations through the release of noradrenaline

We measured the cardiac contractile effects of the sympathomimetic amphetamine-like drug methamphetamine alone and in the presence of cocaine or propranolol in human atrial preparations. For a more comprehensive analysis, we also examined the effects of methamphetamine in preparations from the left and right atria of mice and, for comparison, analyzed the cardiac effects of amphetamine itself. In human atrial preparations, methamphetamine and amphetamine increased the contractile force, the relaxation rate, and the rate of tension development, and shortened the time to maximum tension and the time to relaxation. Likewise, in mice preparations, methamphetamine and amphetamine increased the contractile force in the left atrium and increased the beating rate in the right atrium. The effect in human atrial preparations started at 1 µM, therefore methamphetamine was less effective and potent than isoproterenol in increasing contractile force. These positive inotropic effects of methamphetamine were greatly attenuated by 10 µM cocaine and abolished by 10 µM propranolol. The inotropic effects of methamphetamine in human atrial preparations were associated with, and are believed to be mediated at least in part by, an increase in the phosphorylation state of the inhibitory subunit of troponin. In conclusion, the sympathomimetic central stimulant drug methamphetamine (as well as amphetamine) increased contractile force and protein phosphorylation, presumably through a release of noradrenaline in isolated human atrial preparations. Thus, methamphetamine acts as an indirect sympathomimetic in the human atrium.

C42 OR 1896 EXERTS POSITIVE INOTROPIC EFFECTS IN ISOLATED HUMAN RIGHT ATRIAL MUSCLE

Abstract Levosimendan is approved for use as an intravenous agent to treat temporarily patients with severe systolic heart failure. It is thought, but hitherto never tested in isolated human cardiac tissue, that the main metabolite of levosimendan, called OR 1896, might cause the positive inotropic effect of levosimendan in patients. For comparison, we studied mouse atrial preparations. OR alone (up to 10 µM) failed to increase force of contraction in mouse atrial preparations while in the presence of the phosphodiesterase (PDE) IV–inhibitor rolipram (0.1 µM) profound positive inotropic effects and positive chronotropic effects of OR 1895 were recorded in mouse atrial preparations. In contrast to mouse atrium, we noticed that OR 1896 alone (0.01 µM to 10 µM cumulatively applied) exerted time– and concentration–dependent positive inotropic effects 1 µM increase by 72 ± 14.7%, p&amp;lt;0.05, n=7) and shortened the time of relaxation (by 7 .3 %, p&amp;lt;0.05, n=7) in isolated electrically paced human right atrial muscle preparations (obtained during cardiac surgery), that started at 0.1 µM and plateaued at 1 µM OR 1896. The positive inotropic effect of OR 1896 was antagonized by 10 µM propranolol. The positive inotropic effect of OR 1896 in mouse atrial preparations and human right atrial preparations was less effective than that of 1 µM isoprenaline (human atrium by 154 %). The positive inotropic effect of OR 1896 was diminished by pre–incubation of the samples with the PDE III inhibitor cilostamide (10 µM). EMD 57033 (10 µM), a calcium sensitizer, increased force of contraction further in the additional presence of 1 µM OR 1896. Our data suggest that OR 1896 is indeed a positive inotropic agent in the human heart and that OR 1896 acts mainly as a PDE III – inhibitor because the effect of OR 1896 was attenuated by a β–adrenoceptor antagonist and thus OR 1896 is unlikely to act as a calcium sensitizer in the human heart.

Levosimendan increases the phosphorylation state of phospholamban in the isolated human atrium

Levosimendan (up to 10 µM) given alone failed to increase force of contraction in isolated electrically stimulated (1 Hz) left atrial (LA) preparations from wild-type mice. Only in the additional presence of 0.1 µM rolipram, an inhibitor of the activity of phosphodiesterase IV, levosimendan increased force of contraction in LA and increased the phosphorylation state of phospholamban at amino acid serine 16. Levosimendan alone increased the beating rate in isolated spontaneously beating right atrial preparations from mice and this effect was potentiated by rolipram. The positive inotropic and the positive chronotropic effects of levosimendan in mouse atrial preparations were attenuated by 10 µM propranolol. Finally, we studied the contractile effects of levosimendan in isolated electrically stimulated (1 Hz) right atrial preparations from the human atrium (HAP), obtained during cardiac surgery. We detected concentration-dependent positive inotropic effects of levosimendan alone that reached plateau at 1 µM levosimendan in HAP (n = 11). Levosimendan shortened time of tension relaxation in HAP. Cilostamide (1 µM), an inhibitor of phosphodiesterase III, or propranolol (10 µM) blocked the positive inotropic effect of levosimendan in HAP. Levosimendan (1 µM) alone increased in HAP the phosphorylation state of phospholamban. In conclusion, we present evidence that levosimendan acts via phosphodiesterase III inhibition in the human atrium leading to phospholamban phosphorylation and thus explaining the positive inotropic effects of levosimendan in HAP.

Propranolol Attenuates Late Sodium Current in a Long QT Syndrome Type 3-Human Induced Pluripotent Stem Cell Model.

BackgroundLong QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the SCN5A gene, which encodes the α subunit of the cardiac voltage-gated sodium channel. LQT3 patients present bradycardia and lethal arrhythmias during rest or sleep. Further, the efficacy of β-blockers, the drug used for their treatment, is uncertain. Recently, a large multicenter LQT3 cohort study demonstrated that β-blocker therapy reduced the risk of life-threatening cardiac events in female patients; however, the detailed mechanism of action remains unclear.ObjectivesThis study aimed to establish LQT3-human induced pluripotent stem cells (hiPSCs) and to investigate the effect of propranolol in this model.MethodAn hiPSCs cell line was established from peripheral blood mononuclear cells of a boy with LQT3 carrying the SCN5A-N1774D mutation. He had suffered from repetitive torsades de pointes (TdPs) with QT prolongation since birth (QTc 680 ms), which were effectively treated with propranolol, as it suppressed lethal arrhythmias. Furthermore, hiPSCs were differentiated into cardiomyocytes (CMs), on which electrophysiological functional assays were performed using the patch-clamp method.ResultsN1774D-hiPSC-CMs exhibited significantly prolonged action potential durations (APDs) in comparison to those of the control cells (N1774D: 440 ± 37 ms vs. control: 272 ± 22 ms; at 1 Hz pacing; p < 0.01). Furthermore, N1774D-hiPSC-CMs presented gain-of-function features: a hyperpolarized shift of steady-state activation and increased late sodium current compared to those of the control cells. 5 μM propranolol shortened APDs and inhibited late sodium current in N1774D-hiPSC-CMs, but did not significantly affect in the control cells. In addition, even in the presence of intrapipette guanosine diphosphate βs (GDPβs), an inhibitor of G proteins, propranolol reduced late sodium current in N1774D cells. Therefore, these results suggested a unique inhibitory effect of propranolol on late sodium current unrelated to β-adrenergic receptor block in N1774D-hiPSC-CMs.ConclusionWe successfully recapitulated the clinical phenotype of LQT3 using patient-derived hiPSC-CMs and determined that the mechanism, by which propranolol inhibited the late sodium current, was independent of β-adrenergic receptor signaling pathway.

Beta adrenergic receptor activation inhibits oral cancer migration and invasiveness

ObjectiveThe aim of this study was to verify β2-AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of this receptor in migration and invasion of these neoplastic cells. DesignSCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR. Cell migration and invasion were analyzed by wound healing assay and transwell invasion camera system. Different concentrations (0.1, 1 and 10 μM) of norepinephrine were used to stimulate, and 1 μM propranolol was used to block the beta-adrenergic receptors on cancer cells. Differences in median values of SCC-9 and SCC-25 and β2-AR protein expression were analyzed by Friedman test and in case of significant differences; pairwise comparisons were performed using Bonferroni correction. ResultsThe results showed that the β2-AR gene and protein expression were observed in both oral cancer cell lines. The concentration of 10 μM of norepinephrine significantly inhibited (p ≤ 0.05) migration of SCC-9 and SCC-25 cell lines. Furthermore, there was a significant reduction (p ≤ 0.05) in the effect of norepinephrine on cell migration when the β2-AR was inhibited by propranolol. The blockade by propranolol showed a tendency to reverse the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25. ConclusionsThe use of beta-adrenergic receptor agonists could become an adjuvant therapeutic target in the treatment of this malignancy.

Effect of Tityus serrulatus scorpion venom on isolated jejunum: A very useful tool to study the interaction between neurons in the enteric nervous system

Scorpion envenomation is a public health problem in tropical and subtropical areas. In Brazil, Tityus serrulatus is the biggest cause of accidents with venomous animals. Tityus serrulatus venom causes symptoms related to a great activation of the autonomic system attributed to a massive release of sympathetic and parasympathetic mediators. This effect is attributed to the presence of toxins acting in Na+ and K+ ion channels, leading to an increase in cell excitability. Although gastrointestinal symptoms, like diarrhoea and sialorrhea, is observed in moderate to severe cases, little attention is given in clinical reports. Gastrointestinal motility is controlled by the enteric nervous system which is composed of a wide variety of interconnected neurons that are influenced by the sympathetic and parasympathetic nervous systems. Thus, this work aimed to characterize the effects of Tityus serrulatus venom on sympathetic and parasympathetic neurotransmission of rat jejunum, as well as to investigate possibles effects on other neurons of the enteric nervous system. To this, we verify the effects of Tityus serrulatus venom on the contractility of isolated rat jejunum through organ-bath experiments. We observed that venom can induce both contraction and relaxation. The contraction was partially inhibited by atropine (1 μM) and by suramin (0.1mM) through tetrodotoxin-resistant and sensitive mechanisms. The relaxation was completely inhibited by 3 μM propranolol and partially inhibited by 1 μM phentolamine. Suramin induced a slowing of relaxation curve. Tetrodotoxin completely inhibits the relaxation induced by Tityus serrulatus venom, but the contraction curves were only partially reduced in their initial portion. The final part of the curve was largely enhanced by Tetrodotoxin. Atropine blocks almost completely the contraction curve in the presence of Tetrodotoxin. These results indicate that Tityus serrulatus venom induces the release of both excitatory (predominantly acetylcholine) and inhibitory (mainly noradrenaline) neurotransmitters. The effects of Tityus serrulatus venom on organ contractility was quite complex and seem to derive from a diffuse and nonspecific release of mediators from autonomic and enteric nervous systems. Further investigation of venom action and its isolated toxins can reveal important aspects to deepen our knowledge about the enteric nervous system transmission and the interaction between excitatory and inhibitory mediators as well as the physiological role of Na+ and K+ ion channels in gut motility.

Physiologically Based Pharmacokinetic Modeling of Propranolol in Relation to CYP2D6 Genotypes

Propranolol, used to treat high blood pressure and irregular heart rate, is mainly metabolized by cytochrome P450 2D6 (CYP2D6) to 4‐hydroxypropranolol (4‐OHP) and CYP1A2 to N‐desisoprophylpropranolol (N‐DIP). Physiologically based pharmacokinetic (PBPK) modeling is a mathematical modeling technique for predicting the absorption, distribution, metabolism and excretion (ADME) of synthetic or natural chemical substances in humans and other animal species. We investigated whether the pharmacokinetics of propranolol was altered by the different CYP2D6 genotypes in Korean subjects. Then, through in vitro study of propranolol, the metabolic capacity of propranolol was measured according to the CYP2D6 genotypes. And we developed the PBPK model of propranolol related to CYP2D6 genetic polymorphism. Twenty‐five volunteers were grouped as CYP2D6 *wt ⁄ *wt (*wt=1 or 2, n=7), CYP2D6 *1/ *10 (n=3), CYP2D6 *1 ⁄ *5 (n=4) and CYP2D6 *10 ⁄ *10 (n=11) according to their genotypes. Propranolol hydrochloride 40 mg (Pranol®) was administered orally once to each subject in these four groups. The CYP2D6 *1, CYP2D6 *10 and CYP1A2 *1 were incubated with 0.5–100 μM propranolol for 60 min at 37 □. Then the metabolites were extracted, and the concentration was analyzed by liquid chromatography‐mass spectrometry. PBPK modeling of propranolol was developed and optimized using PK‐sim® software. And, validation of PBPK model was conducted by comparing the predicted values with observed values from comparison the pharmacokinetic studies. In clinical data, AUCend, AUCinf, Cmax and oral clearance of propranolol were significantly different in CYP2D6 genetic polymorphism (P&lt;0.005, respectively). Depending on the physico‐chemical parameters and ADME of each genotype, PBPK model of propranolol was developed. For metabolism, input values of in vitro Vmax of 4‐OHP in the presence of recombinant enzyme for CYP2D6 *1, CYP2D6 *10, and CYP1A2 *1 were 2.41, 0.344 and 0.383 pmol/min/pmol, respectively. And, input values of Km of 4‐OHP for CYP2D6 *1, CYP2D6 *10, and CYP1A2 *1 were 2.852, 8.914 and 6.95 μM, respectively. Input values of in vitro Vmax of N‐DIP in the presence of recombinant enzyme for CYP2D6 *1, CYP2D6 *10, and CYP1A2 *1 were 9.782, 7.891 and 11.18 pmol/min/pmol, respectively. And, input values of Km of N‐DIP for CYP2D6 *1, CYP2D6 *10, and CYP1A2 *1 were 162.8, 339.1 and 158.4 μM, respectively. The AUCinf mean of simulated PBPK model were 111.77, 184.74, 296.13, 370.85 ng·h/ml in CYP2D6 *wt ⁄ *wt, CYP2D6 *1 / *10, CYP2D6 *1 ⁄ *5 and CYP2D6 *10 ⁄ *10, respectively. The developed PBPK model of propranolol successfully described the pharmacokinetics of each CYP2D6 genotype group and its simulated values were within acceptance criterion (99.998% confidence interval). These results will be beneficial in prescribing the appropriate dosage of propranolol considering inter‐individual differences through this mechanical approach. In addition, the PBPK modeling of propranolol in relation to CYP2D6 genotypes will be applicable to the treatment of various ethnic groups, ages, and patients. The observed and predicted pharmacokinetics for propranolol with acceptance criterion range in CYP2D6 genotype groups. Genotype Cmax (ng/mL) AUCend (ng*h/mL) AUCinf (ng*h/mL) Tmax (h) T1/2 (h) CYP2D6*wt/*wt (n=7) Observed 21.93±5.74 132.2±41.39 150.07±40.34 1.79±0.27 4.04±0.94 Predicted 30.70 111.53 111.77 1.90 2.83 Aceeptance range 14.49–33.19 80.79–216.30 98.09–229.60 1.41–2.27 2.80–5.84 CYP2D6*1/*10 (n=3) Observed 32.30±5.92 149.28±62.51 165.18±71.82 1.50±0.50 3.27±0.61 Predicted 34.74 181.01 184.74 1.95 4.37 Aceeptance range 20.66–50.51 55.55–401.16 59.36–459.66 0.68–3.33 2.08–5.14 CYP2D6*1/*5 (n=4) Observed 43.75±16.07 245.37±100.75 255.58±99.22 1.75±0.29 3.72±1.14 Predicted 51.88 286.96 296.13 1.95 5.02 Aceeptance range 20.51–93.32 105.85–568.78 115.06–567.70 1.23–2.48 1.96–7.06 CYP2D6*10/*10 (n=11) Observed 48.77±17.07 339.82±106.89 360.32±110.67 2.27±0.82 4.68±1.38 Predicted 62.70 357.72 370.85 2.00 5.14 Aceeptance range 31.51–75.48 229.04–504.19 245.02–529.88 1.45–3.56 3.23–6.78

Insulin-like growth factor 2 promotes the adipogenesis of hemangioma-derived stem cells.

Infantile hemangioma (IH), which is the most common tumor in infants, is characterized by rapid proliferation followed by spontaneous regression into fibro-fatty tissue in childhood. However, its specific mechanism has not been clarified. Our previous studies showed that insulin-like growth factor 2 (IGF-2) is increased in the proliferative phase of IH, which is deemed to form from hemangioma-derived stem cells (HemSC). However, it remains unclear whether IGF-2 can promote the adipogenic differentiation of HemSCs and the signaling mechanisms involved require further elucidation. In the present study, CCK-8 assay was used to detect the effect of different concentrations of IGF-2 on the proliferation of HemSCs. Immunohistochemistry was applied to observe the expression of IGF-2 and its receptors in cells. Oil red o-staining of adipogenesis was conducted after cells recevied no treatment or were induced with IGF-2 or IGF-2 plus OSI-906 for 10 days. Cells were cultured in EGM-2/FBS-10% alone or containing IGF-2, IGF-2 plus OSI-906 or IGF-2 plus LY294002 and the protein expression of C/EBPα, C/EBPβ, PPARγ, adiponectin, p-AKT and total AKT was determined using western blot analysis. In another experiment, cells were treated with 25, 50 or 100 μM propranolol, or vehicle. C/EBPα, C/EBPβ, PPARγ and IGF-2 were analyzed using western blot analysis or reverse transcription-quantitative polymerase chain reaction. Results indicated that IGF-2 significantly promoted the cell proliferation and lipid accumulation of HemSCs. The expression of phosphorylated AKT (p-AKT), C/EBPα, C/EBPβ, PPARγ and adiponectin was increased in IGF-2-treated HemSCs culture, whereas these changes were repressed by the inhibition of either the IGF-1 receptor (IGF-1R) or phosphoinositide 3-kinase (PI3K). Our previous research showed that propranolol accelerated adipogenesis in HemSCs and induced the upregulation of IGF-2. The results of the present study indicate that IGF-2 is able to accelerate adipogenesis, and the propranolol-induced promotion of dysregulated adipogenesis may be mediated by the IGF-2 via IGF-1R and PI3K pathways.

Trends in Severe Obesity Among 23 Million U.S. Children Aged 2–4 Years Who Enrolled in WIC — United States, 2000–2014

The aim of this study was to verify β2-AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of this receptor in migration and invasion of these neoplastic cells.SCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR. Cell migration and invasion were analyzed by wound healing assay and transwell invasion camera system. Different concentrations (0.1, 1 and 10 μM) of norepinephrine were used to stimulate, and 1 μM propranolol was used to block the beta-adrenergic receptors on cancer cells. Differences in median values of SCC-9 and SCC-25 and β2-AR protein expression were analyzed by Friedman test and in case of significant differences; pairwise comparisons were performed using Bonferroni correction.The results showed that the β2-AR gene and protein expression were observed in both oral cancer cell lines. The concentration of 10 μM of norepinephrine significantly inhibited (p ≤ 0.05) migration of SCC-9 and SCC-25 cell lines. Furthermore, there was a significant reduction (p ≤ 0.05) in the effect of norepinephrine on cell migration when the β2-AR was inhibited by propranolol. The blockade by propranolol showed a tendency to reverse the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25.The use of beta-adrenergic receptor agonists could become an adjuvant therapeutic target in the treatment of this malignancy.

Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min−1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.—Gherbi, K., May, L. T., Baker, J. G., Briddon, S. J., Hill, S. J. Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

Inhibition of a TREK-like K+ channel current by noradrenaline requires both β1- and β2-adrenoceptors in rat atrial myocytes.

AimsNoradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss).Methods and resultsRat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca2+ current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K+ channel. The effect of noradrenaline on IKss was abolished by combined β1-/β2-adrenoceptor antagonism (1 µM propranolol or 10 µM β1-selective atenolol and 100 nM β2-selective ICI-118,551 in combination), but not by β1- or β2-antagonist alone. The action of noradrenaline could be mimicked by β2-agonists (zinterol and fenoterol) in the presence of β1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by β1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined β1-/β2-antagonism, but not by atenolol alone.ConclusionNoradrenaline inhibits a rat atrial TREK-like K+ channel current via a PTX-sensitive mechanism involving co-operativity of β1-/β2-adrenoceptors that contributes to atrial APD prolongation.

Propranolol does not increase the contractile response to epinephrine in endothelium-intact aorta from renal hypertensive rat

Renal hypertension (2K-1C) is characterized by the increased sympathetic activity and oxodative stress induced by angiotensin II. Therefore, the epinephrine plasma levels can potentiate the contractile response in 2K-1C rat aorta. This study aimed to evaluate the effect of inhibition of β-adrenoceptor (β-AR) activation on the contractile response induced by epinephrine in intact endothelium (E+) and in denuded aorta (E−) of 2K-1C rats compared to normotensive sham-operated rats (2K). All the procedures were performed in accordance with the standards and policies of Animal Care and Use Committee of the University of São Paulo. Male rats (180–200 g) were anesthetized with tribromoethanol and after a midline laparotomy a silver clip was placed around the left renal artery. Normotensive two-kidney rats (2K) were only submitted to laparotomy. The systolic blood pressure (SBP) was measured by an indirect tail-cuff method 6 weeks after surgery. Rats were considered to be hypertensive when systolic arterial pressure was higher than 160 mm Hg. Rats were killed and the aortic rings were isolated. Concentration–effect curves were constructed for epinephrine in E+ and E− aortas in the absence (Control) or after incubation with 10 μM propranolol (P+) for 20 min. The maximal effect (ME, in grams of tension) and the potency (pD2) were evaluated. Propranolol did not change the contractile response induced by epinephrine in aortas E− from 2K rats (Control, ME: 2.9 ± 0.2 g; pD2: 7.74 ± 0.09; n = 9) and P+ (ME: 3.7 ± 0.3 g; pD2: 7.48 ± 0.19; n = 4) and 2K-1C (Control, ME: 2.1 ± 0.1 g; pD2: 7.92 ± 0.10; n = 7) and P+ (ME: 2.2 ± 0.2 g; pD2: 8.44 ± 0.78; n = 5). In E+ aorta, propranolol increased the ME to epinephrine in 2K rat aortas from 2.6 ± 01 g; n = 11 to 3.1 ± 0.2 g; n = 6) (P <0.01) and it reduced the pD2 values in 2K-1C rat aortas from 7.37 ± 0.16; n = 9 to 6.70 ± 0.22; n = 4) (P <0.05). Our results indicate that the contractile response induced by epinephrine involves the activation of β-AR in the endothelial cells of 2K rat aortas that leads to a lower contractile responses of 2K rat aorta but not in 2K-1C hypertensive rat aorta. Supported by FAPESP and CNPq .

α1A‐Adrenergic regulation of inhibition in the olfactory bulb

By regulating inhibition at dendrodendritic synapses between mitral and granule cells (GCs), noradrenergic neurons extending from the brainstem provide an input essential for odour processing in the olfactory bulb (OB). In the accessory OB (AOB), we have recently shown that noradrenaline (NA) increases GABA inhibitory input on to mitral cells (MCs) by exciting GCs. Here, we show that GCs in the main OB (MOB) exhibit a similar response to NA, indicating a common mechanism for noradrenergic regulation of GCMC inhibition throughout the OB. In GCs of the MOB, NA (10 μM) produced a robust excitatory effect that included a slow afterdepolarization that followed a train of action potentials evoked by a current stimulus. The depolarization and slow afterdepolarization in GCs were blocked by the α1A-adrenergic receptor (AR) selective antagonist WB 4101 (30 nm) and mimicked by the α(1A)-AR selective agonist A 61603 (1 μM). In recordings from MCs, A 61603 (30 nm-1 μM) produced a sizeable increase in the frequency of spontaneous and miniature IPSCs, an effect completely abolished by the GABAA receptor antagonist gabazine (5 μM). Likewise, activation of β-ARs increased the frequency of spontaneous IPSCs; however, this effect was smaller and confined to the first postnatal weeks. NA enhanced inhibition in MCs across a broad concentration range (0.1-30 μM) and its effects were completely abolished by a mixture of α1- and β-AR antagonists (1 μM prazosin and 10 μM propranolol). Furthermore, the general α2-AR agonist clonidine (10 μM) failed to affect sIPSC frequency. Thus, the NA-mediated increase in GCMC inhibition in the OB results mostly from activation of the α1A-AR subtype.

Propranolol Accelerates Adipogenesis in Hemangioma Stem Cells and Causes Apoptosis of Hemangioma Endothelial Cells

Infantile hemangiomas can cause significant morbidity during proliferation, yet there is no U.S. Food and Drug Administration-approved treatment. They are believed to form from hemangioma stem cells, which differentiate toward a hemangioma endothelial cell phenotype. Recently, propranolol has demonstrated effectiveness in treating complicated infantile hemangiomas. The authors hypothesize that propranolol facilitates their involution by altering cellular behavior in both hemangioma endothelial and stem cells. Hemangioma endothelial and stem cells were isolated from resected infantile hemangioma specimens. Cells were treated with 100 μM propranolol for 48 hours, and apoptosis was determined by the presence of annexin V antibody. Proliferation of stem and endothelial cells was assessed after treatment with 50 or 100 μM propranolol or vehicle, for 72 and 96 hours, respectively. Adipogenesis was induced in stem cells with and without propranolol. Pro-adipogenic genes PPARδ, PPARγ, C/EBPα, C/EBPβ, C/EBPδ, RXRα, and RXRγ were analyzed by quantitative polymerase chain reaction. Annexin V levels were increased in propranolol-treated endothelial cells but not in stem cells. Proliferation of stem and endothelial cells was inhibited by propranolol in a dose-dependent manner. Propranolol-treated stem cells demonstrated accelerated adipogenesis when compared with untreated controls. Transcript levels of C/EBPβ (p < 0.05), RXRγ (p < 0.05), and PPARγ (p < 0.02) were significantly increased when treated with 50 or 100 μM propranolol; and C/EBPδ (p < 0.05), RXRα (p < 0.05), and PPARδ (p < 0.01) transcripts were increased when treated with 100 μM propranolol. C/EBPα transcript levels remained unchanged at either dose. Propranolol increased apoptosis of hemangioma endothelial cells, but not stem cells, and accelerated adipogenesis of hemangioma stem cells. Thus, propranolol likely accelerates involution to fibrofatty residuum.