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Probucol is an antihyperlipidemic drug with potent antioxidant properties. Oxidative stress plays an important role in the pathogenesis of diabetic retinopathy. In this study, we aimed to investigate the protective effects of probucol against diabetes-induced retinal vascular dysfunction in a rat model of diabetes. Diabetes was induced by a combination of streptozotocin treatment and D-glucose feeding, and retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles. The vasodilator effect of salbutamol, a β-adrenoceptor agonist, on retinal arterioles was significantly diminished 2 weeks after the induction of diabetes. In non-diabetic rats, vasodilator responses to salbutamol were significantly reduced after an intravitreal injection of iberiotoxin, a blocker of large-conductance K (BK) channels. However, this effect was not observed in diabetic rats. Probucol had no significant effect on salbutamol-induced changes in diameter of retinal arterioles in non-diabetic rats, whereas it could prevent the attenuation of retinal vasodilator response to salbutamol in diabetic rats. These results suggest that the reduced function of BK channels is involved in the attenuation of β-adrenoceptor-mediated retinal vasodilation in diabetic rats. Probucol preserves the BK channel function in retinal arterioles under diabetic conditions; therefore, it may show beneficial effects on diabetic retinopathy by preventing or slowing the impairment of the retinal circulation in patients with diabetes mellitus. [ABSTRACT FROM AUTHOR]

We have previously shown that the natriuretic peptide receptor B (NPR-B) agonist C-type natriuretic peptide (CNP) enhances cyclic adenosine 3´,5´-monophosphate (cAMP)-mediated signaling in failing hearts, through cyclic guanosine 3´,5´-monophosphate (cGMP)-mediated phosphodiesterase (PDE) 3 inhibition. As several signaling pathways are importantly changed in failing hearts, it could not be taken for granted that this crosstalk would be the same in non-failing hearts. Thus, we wanted to clarify to which extent this effect of CNP occurred also in non-failing hearts. Inotropic and lusitropic responses were measured in muscle strips and cGMP levels, localized cAMP levels, cAMP-PDE activity and mRNA levels were analyzed in isolated cardiomyocytes from left ventricles of non-failing and failing rat hearts. CNP increased cGMP and enhanced β 1 - and β 2 -adrenoceptor-mediated inotropic and β 1 -adrenoceptor-mediated lusitropic responses, in non-failing and failing hearts. The NPR-A agonist brain natriuretic peptide (BNP) increased cGMP, but did not affect inotropic or lusitropic responses, indicating different compartmentation of cGMP from the two natriuretic peptide receptors. cAMP-PDE activity of PDE3 was concentration-dependently inhibited by cGMP with the same potency and to the same extent in non-failing and failing cardiomyocytes. CNP enhanced β 1 -adrenoceptor-induced cAMP increase in living cardiomyocytes in the absence, but not in the presence of a PDE3 inhibitor indicating involvement of PDE3. In summary, CNP sensitizes cAMP-mediated signaling in non-failing as in failing hearts, via NPR-B-mediated increase of cGMP that inhibits the cAMP-PDE activity of PDE3. [ABSTRACT FROM AUTHOR]

The sympathetic nervous system play a pivotal role in bone remodeling through β-adrenoceptor (β-AR). However, it is not well documented whether the β-adrenoceptor pathway has the potential to influence osteocytes. In this study, cell viability, the expression of β-AR subtypes, enzymes of catecholamine synthesis or degradation, bone-related gene and protein in osteocytic MLO-Y4 cells were investigated by β-adrenergic stimulation. Isoproterenol (ISO) promoted RANKL to OPG expression in osteocytes, as well as osteoclasts formation in osteocytes-RAW264.7 cell co-cultures but not RAW264.7 cell monoculture. The ISO-stimulated effect was enhanced in β1-AR antagonist pretreatment, but was rescued by blocking β2-AR. The results indicate that β1-and β2-AR play reciprocal roles on MLO-Y4 cells in the regulation of osteoclastogenesis, and osteocyte β-adrenergic signaling might be a new valuable therapy for bone disease. [ABSTRACT FROM AUTHOR]

Lead is considered a causative factor for hypertension and other cardiovascular diseases. Aims To investigate the effects of sub-chronic lead exposure on blood pressure reactivity and cardiac β 1 -adrenoceptor activity and to evaluate whether the effects found in vitro are similar to those found in vivo . Main methods Male Wistar rats were randomly distributed into two groups: control rats (Ct) and rats administered drinking water containing 100 ppm lead (Pb) for 30 days. Key findings Blood pressure in the Pb rats increased starting from the first week of treatment until the end of the study [systolic blood pressure, Ct: 122 ± 4 vs . Pb: 143 ± 3 mmHg; diastolic blood pressure, Ct: 63 ± 4 vs. Pb: 84 ± 4 mmHg]. The heart rate was also increased (Ct: 299 ± 11 vs. Pb: 365 ± 11 bpm), but the pressure reactivity to phenylephrine was decreased. Losartan and hexamethonium exhibited a greater reduction in blood pressure of Pb rats than in the Ct rats. Isoproterenol increased the left ventricular systolic and end-diastolic pressure, and heart rate only in Ct rats, suggesting that lead induced β 1 -adrenoceptor downregulation. Indomethacin reduced the blood pressure and heart rate in the Pb rats, suggesting the involvement of cyclooxygenase-derived products (which are associated with reduced nitric oxide bioavailability) in this process. Significance These findings offer further evidence that the effects of sub-chronic lead exposure in vitro can be reproduced in vivo —even at low concentrations—thus triggering mechanisms for the development of hypertension. Therefore, lead should be considered an environmental risk factor for cardiovascular disease. [ABSTRACT FROM AUTHOR]

The influence of cell confluence on the β-adrenoceptor (β-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·10 3 cells/cm 2 ) or high density (HD: 3·10 4 cells/cm 2 ) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. β-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15 s) of the β-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100 nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100 μM) induced a similar saturating response in both LD and HD cells. A β 1 -AR antagonist (CGP 20712A, 100 nM) reduced the Iso (100 nM) response in HD but not LD cells, whereas a β 2 -AR antagonist (ICI 118,551, 5 nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [ 125 I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β 1 - and β 2 -ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β 2 -ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP] i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10 μM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1 μM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the β-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both β 1 - and β 2 -AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β 2 -AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the β 1 -AR, a lower cAMP-PDE activity and a higher basal [cAMP] i . This study highlights the critical role of the cellular environment in controlling the vascular β-AR signalling. [ABSTRACT FROM AUTHOR]

Suo Quan Wan (SQW) has been used to treat lower urinary tract symptoms (LUTS) in elderly patients for hundreds of years in China. β-adrenoceptors (β-ARs), particularly β3-adrenoceptor (β3-AR), was reported to be important in the bladder dysfunction of the elderly. The present study was conducted to explore the effect of β-AR, and particularly the β3-adrenoceptor, in aging rat bladder function in vitro and to test the therapeutic effect of SQW on LUTS in an aging rat model based on the β3-adrenoceptor. Briefly, the bladder detrusor muscles of young (age, 3 months) and aging (age, 15 months) female rats were separated. A β-AR non-selective agonist, isoprenaline (ISO), subtype β3-AR agonist (BRL37344A) and β3-AR antagonist (SR59230A) were used to define the tension change of detrusor muscles between young and aging rats in vitro. For blank controls, 12 young rats were marked, and 48 aging female rats were randomly divided into four groups as follows: Model, SQW high, SQW middle and SQW low. Following oral administration of SQW for 6 weeks in aging rats, urodynamic and bladder detrusor tests were used to evaluate the therapeutic effect of SQW. The expression of β3-AR mRNA was investigated using reverse transcription-quantitative polymerase chain reaction. Using ISO and BRL37344A in vitro, maximum relaxation (Emax), intrinsic activity (IA), and log (50% effective concentration) (PD2) were significantly decreased in aging rats compared with that in young rats (P<0.05). Significant changes were also observed in the β3-AR antagonist experiment, which blocked ISO-induced relaxation, with significant decreases observed in Emax, IA and PD2, and a significant increase observed in PA2 for the aging rats compared with the young controls (P<0.05). SQW was demonstrated to enhance bladder control, storage and contraction ability. Furthermore, SQW was able to increase the sensitivity and expression of β3-AR in an aging rat. In conclusion, the decrease in β3-AR sensitivity in aging rats and the expression resulted in bladder detrusor dysfunction. In addition, the therapeutic effect of SQW against LUTS relies on the former's effect on the urethral sphincter, bladder detrusor and β3-AR. [ABSTRACT FROM AUTHOR]

Purpose The aim of this study is to evaluate the effect of retigabine on the smooth muscle response to acetylcholine, adrenaline, α-and β-adrenoceptor agonists. Methods We studied the change in the spontaneous smooth muscle contraction of guinea pig gastric corpus strips before and after 20-min treatment with 2 μM retigabine. We also evaluated the effect of retigabine on the smooth muscle response to 10 μM acetylcholine, 1 and 10 μM adrenaline, 1 μM methoxamine, 0.1 μM p-iodoclonidine and 10 μM isoproterenol. Results We observed a significant reduction in the effects of all studied mediators and agonists when they were added to organ baths in the presence of retigabine. Retigabine diminished the effect of acetylcholine on the spontaneous smooth muscle activity. The effect was fully antagonized by XE-991 (Kv7 channel blocker), which supports our hypothesis about the role of KCNQ channels in the registered changes. The increase in the contraction force after adding of 1 μM adrenaline, methoxamine, and 0.1 μM p-iodoclonidine was also significantly smaller in presence of retigabine. However, comparing the effect of 10 μM adrenaline on the contractility before and after treatment with retigabine, we observed increased contractility when retigabine was present in the organ baths. Conclusion A possible explanation for the observed diminished effects of mediators and receptor agonists is that the effect of retigabine on smooth muscle contractility is complex. The membrane hyperpolarization, the interaction between Kv7 channels and adrenoceptors, and the influence on signaling pathways may contribute to the summary smooth muscle response. [ABSTRACT FROM AUTHOR]

Background & Purpose: Hydrogen (H2) has been shown to have a strong antioxidant effect on preventing oxidative stress-related diseases. The goal of the present study is to determine the pharmacodynamics of H2 in a model of isoproterenol (ISO)-induced cardiac hypertrophy.Methods: Mice (C57BL/6J; 8-10 weeks of age) were randomly assigned to four groups: Control group (n=10), ISO group (n=12), ISO plus H2 group (n=12), H2 group (n=12). Mice received H2 (1ml/100g/day, intraperitoneal injection) for 7 days before ISO (0.5mg/100g/day, subcutaneous injection) infusion, and then administered ISO with or without H2 for another 7 days. Then, cardiac function was evaluated by echocardiography. Cardiac hypertrophy was reflected by heart weight/body weight, gross hearts and heart sections stained with H&E, and relative ANP and BNP mRNA levels. Cardiac reactive oxygen species (ROS), 3-nitrotyrosine and p67 (phox) levels were analyzed by Dihydroethidium staining, immunohistochemistry and western blotting, respectively. For in vitro study, H9c2 cardiomyocytes were pretreated with hydrogen rich medium for 30 minutes, and then administrated with ISO (10 μM) for the indicated time. The medium and ISO were re-changed every 24 hours. Cardiomyocyte surface areas, relative ANP and BNP mRNA levels, the expression of 3-nitrotyrosine, and the dissipation of mitochondrial membrane potential (MMP) were examined. Moreover, the expression of ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK were measured by western blotting both in vivo and in vitro.Results: Intraperitoneal injection of hydrogen prevents cardiac hypertrophy and improves cardiac function in ISO-infused mice. Hydrogen rich medium blocks ISO-mediated cardiomyocytes hypertrophy in vitro. Hydrogen blocked the excessive expression of NADPH oxidase and the accumulation of ROS, attenuated the decrease of MMP, and inhibited ROS-sensitive ERK1/2, p38, JNK1/2 signaling pathways.Conclusions: H2 inhibited ISO-induced cardiac/cardiomyocytes hypertrophy both in vivo and in vitro, and improved the impaired left ventricular function. H2 exerts its protective effects partially through blocking ROS-sensitive ERK1/2, p38, JNK1/2 signaling pathways.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca2+-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were 2-3 fold smaller than adult CM, independently of culture format (capacitance ML 45±1 pF (n=289), EHT 45±1 pF (n=460), atrial CM 87±3 pF (n=196), ventricular CM 126±8 pF (n=50)). Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10±1 pA/pF (n=207) for ML and 12±1 pA/pF (n=361) for EHT and was larger than in adult CM (7±1 pA/pF (p

BackgroundPropofol may decrease myocardial contractility via actions on the β-adrenoceptor-mediated signal transduction. The aim of this study was to evaluate the effect of propofol via β-adrenoceptor-mediated signal transduction by measuring the tissue levels of cAMP (cyclic adenosine monophosphate).MethodsThe effects of propofol on β-adrenoceptor mediated cascades were measured with cAMP concentrations, which were stimulated by agonists (l-isoproterenol, GTPγS, and forskolin) of each step of β-adrenoceptor-mediated cascades.ResultsWhile the production of cAMP stimulated by isoproterenol, GTPγS, or forskolin are increased (P < 0.05), application of each concentration of propofol (0.1, 1, 10, 100 µM) did not alter the levels of cAMP.ConclusionsConsidering that propofol did not alter the tissue cAMP levels when stimulated by isoproterenol, GTPγS, and forskolin, propofol appears to have no effect on the β-adrenoceptor signaling pathway in guinea pig ventricular myocardium.

Early odor preference learning in rodents occurs within a sensitive period (≤postnatal day (P)10-12), during which pups show a heightened ability to form an odor preference when a novel odor is paired with a tactile stimulation (e.g. stroking). Norepinephrine (NE) release from the locus coeruleus during stroking mediates this learning. However, in older pups, stroking loses its ability to induce learning. The cellular and circuitry mechanisms underpinning the sensitive period for odor preference learning is not well understood. We first established the sensitive period learning model in mice - odor paired with stroking induced odor preference in P8 but not P14 mice. This learning was dependent on NE-β-adrenoceptors as it was prevented by propranolol injection prior to training. We then tested whether there are developmental changes in pyramidal cell excitability and NE responsiveness in the anterior piriform cortex (aPC) in mouse pups. Although significant differences of pyramidal cell intrinsic properties were found in two age groups (P8-11 and P14+), NE at two concentrations (0.1 and 10 μM) did not alter intrinsic properties in either group. In contrast, in P8-11 pups, NE at 0.1 μM presynaptically decreased miniature IPSC and increased miniature EPSC frequencies. These effects were reversed with a higher dose of NE (10 μM), suggesting involvement of different adrenoceptor subtypes. In P14+ pups, NE at higher doses (1 and 10 μM) acted both pre- and postsynaptically to promote inhibition. These results suggest that enhanced synaptic excitation and reduced inhibition by NE in the aPC network may underlie the sensitive period.

Key points β-Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved., Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in β-adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol., We show that while NOS contributes to β-adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation., We also show that combined inhibition of NOS and COX augments β-adrenergic sweating, These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of β-adrenergic receptors in the skin., Abstract β-Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to β-adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to β-adrenergic sweating. In 10 young males (25 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10 m m Nω-nitro- l-arginine ( l-NNA), a non-specific NOS inhibitor, (3) 10 m m ketorolac, a non-specific COX inhibitor, or (4) a combination of l-NNA and ketorolac. All sites were co-administered with a high dose of isoproterenol (100 μ m) for 3 min to maximally induce β-adrenergic sweating (β-adrenergic sweating is significantly blunted by subsequent activations). Approximately 60 min after the washout period, three incremental doses of isoproterenol were co-administered (1, 10 and 100 μ m each for 25 min). Increases in CVC induced by the first and second 100 μ m isoproterenol were attenuated by l-NNA alone, and those in response to all doses of isoproterenol were reduced by l-NNA with co-infusion of ketorolac (all P ≤ 0.05). Ketorolac alone augmented increases in CVC induced by 10 μ m and by the second 100 μ m isoproterenol (both P ≤ 0.05). While isoproterenol-induced sweating was not affected by the separate administration of l-NNA or ketorolac (all P > 0.05), their combined administration augmented sweating elicited by the first 3 min of 100 μ m isoproterenol ( P = 0.05). We show that while NOS contributes to β-adrenergic cutaneous vasodilatation, COX restrains the vasodilatation. Finally, combined inhibition of NOS and COX augments β-adrenergic sweating. [ABSTRACT FROM AUTHOR]

Summary Spike timing-dependent synaptic plasticity (STDP) serves as a key cellular correlate of associative learning, which is facilitated by elevated attentional and emotional states involving activation of adrenergic signaling. At cellular levels, adrenergic signaling increases dendrite excitability, but the underlying mechanisms remain elusive. Here we show that activation of β2-adrenoceptors promoted STD long-term synaptic potentiation at mouse hippocampal excitatory synapses by inactivating dendritic Kv1.1-containing potassium channels, which increased dendrite excitability and facilitated dendritic propagation of postsynaptic depolarization, potentially improving coincidental activation of pre- and postsynaptic terminals. We further demonstrate that adrenergic modulation of Kv1.1 was mediated by the signaling scaffold SAP97, which, through direct protein-protein interactions, escorts β2 signaling to remove Kv1.1 from the dendrite surface. These results reveal a mechanism through which the postsynaptic signaling scaffolds bridge the aroused brain state to promote induction of synaptic plasticity and potentially to enhance spike timing and memory encoding. [ABSTRACT FROM AUTHOR]

We have shown previously that the cannabinoid receptor agonist CP55940 microinjected into the paraventricular nucleus of the hypothalamus (PVN) of urethane-anaesthetized rats induces depressor and pressor cardiovascular effects in the absence and presence of the CB antagonist AM251, respectively. The aim of our study was to examine whether the hypotension and/or hypertension induced by CP55940 given into the PVN results from its influence on glutamatergic and GABAergic neurotransmission. CP55940 was microinjected into the PVN of urethane-anaesthetized rats twice (S and S, 20 min apart). Antagonists of the following receptors, NMDA (MK801), β-adrenergic (ICI118551), thromboxane A-TP (SQ29548), angiotensin II-AT (losartan) or GABA (bicuculline), or the NO synthase inhibitor L-NAME were administered intravenously 5 min before S alone or together with AM251. The CP55940-induced hypotension was reversed into a pressor response by AM251, bicuculline and L-NAME, but not by the other antagonists. The CP55940-induced pressor effect examined in the presence of AM251 was completely reversed by losartan, reduced by about 50-60 % by MK801, ICI118551 and SQ29548, prevented by bilateral adrenalectomy but not modified by bicuculline and L-NAME. Parallel, but smaller, changes in heart rate accompanied the changes in blood pressure. The bi-directional CB receptor-mediated cardiovascular effects of cannabinoids microinjected into the PVN of anaesthetized rats depend on stimulatory glutamatergic and inhibitory GABAergic inputs to the sympathetic tone; the glutamatergic input is related to AT, TP and β-adrenergic receptors and catecholamine release from the adrenal medulla whereas the GABAergic input is reinforced by NO. [ABSTRACT FROM AUTHOR]

Background/Aim: The nocturnal production of melatonin by the pineal gland is triggered by sympathetic activation of adrenoceptors and may be modulated by immunological signals. The effect of glucocorticoids on nocturnal melatonin synthesis is controversial; both stimulatory and inhibitory effects have been reported. During pathophysiological processes, an increased sympathetic tonus could result in different patterns of adrenoceptor activation in the pineal gland. Therefore, in this investigation, we evaluated whether the pattern of adrenergic stimulation of the pineal gland drives the direction of the glucocorticoid effect on melatonin production. Methods: The corticosterone effect on the pineal hormonal production induced by ß-adrenoceptor or ß+a 1-adrenoceptor activation was evaluated in cultured glands. We also investigated whether the in vivo lipopolysaccharide (LPS)-induced inhibition of melatonin is dependent on the interaction of glucocorticoids and the a1-adrenoceptor in adrenalectomized animals and on the in vivo blockade of glucocorticoid receptors (GRs) or the a1-adrenoceptor. Results: Corticosterone potentiated ß-adrenoceptor-induced pineal melatonin synthesis, whilst corticosterone-dependent inhibition was observed when melatonin production was induced by ß+a 1-adrenoceptors agonists. The inhibitory effect of corticosterone is mediated by GR, as it was abolished in the presence of a GR antagonist. Moreover, LPS-induced reduction in melatonin nocturnal plasma content was reversed by adrenalectomy and by antagonizing GR or a1-adrenoceptors. Conclusions: The dual effect of corticosterone on pineal melatonin synthesis is determined by the activation pattern of adrenoceptors (ß or ß+a1) in the gland during GR activation, suggesting that increased activation of the sympathetic system and the hypothalamic-pituitary-adrenal axis are necessary for the control of melatonin production during defense responses. [ABSTRACT FROM AUTHOR]

Objective To evaluate whether active immunization producing β 1 - or β 3 -antibodies (β 1 -ABs and β 3 -ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA). Design and method Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of β 1 - and β 3 -adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various β-AR agonists, phenylephrine and KCl. Results The immunizations producing functional β 1 -ABs and β 3 -ABs did not affect the SBP. However, in TA from β 1 -AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with β 3 -AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with β 1 - or β 3 -peptides, relaxations induced by the various β-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. Conclusions Our study shows that β 1 - and β 3 -ABs can affect vascular reactivity. β 1 -ABs would have a pathogenic action whereas β 3 -ABs would have a beneficial effect on aorta reactivity. [ABSTRACT FROM AUTHOR]

Cardiac resistance against acute ischemia/reperfusion (I/R) injury can be enhanced by adaptation to chronic intermittent hypoxia (CIH), but the changes at the molecular level associated with this adaptation are still not fully explored. Phospholipase A (PLA) plays an important role in phospholipid metabolism and may contribute to membrane destruction under conditions of energy deprivation during I/R. The aim of this study was to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLAα (cPLAα) and its phosphorylated form (p-cPLAα), as well as other related signaling proteins in the left ventricular myocardium of adult male Wistar rats. Adaptation to CIH increased the total content of cPLAα by 14 % in myocardial homogenate, and enhanced the association of p-cPLAα with the nuclear membrane by 85 %. The total number of β-adrenoceptors (β-ARs) did not change but the β/β ratio markedly increased due to the elevation of β-ARs and drop in β-ARs. In parallel, the amount of adenylyl cyclase decreased by 49 % and Gα proteins increased by about 50 %. Besides that, cyclooxygenase 2 (COX-2) and prostaglandin E (PGE) increased by 36 and 84 %, respectively. In parallel, we detected increased phosphorylation of protein kinase Cα, ERK1/2 and p38 (by 12, 48 and 19 %, respectively). These data suggest that adaptive changes induced in the myocardium by CIH may include activation of cPLAα and COX-2 via β-AR/G-mediated stimulation of the ERK/p38 pathway. [ABSTRACT FROM AUTHOR]

Context:Folium Eriobotryae(FE), the dry leaf ofEriobotrya japonica(Thunb.) Lindl. (Rosaceae), has been widely used to treat respiratory disorders. Objective:To examine the bronchodilatory activity of FE and the potential mechanisms involved. Materials and methods:The effects of ethyl acetate fraction of FE (EFE) (0.05–0.3 mg/mL) on the isolated tracheal strips, and ursolic acid (UA) (5–30 μg/mL) that was the main constituent of EFE, were testedin vitro. Meanwhile, acetylcholine (Ach) and histamine (His)-induced bronchospasm were conductedin vivoin guinea pig. Furthermore, mechanisms of relaxant effects of EFE and UA were evaluated in the absence and presence of specific inhibitors. Results:Within vitrostudies, the contractile response evoked by Ach or His (EC50 = 0.21 and 0.16 mg/mL) was decreased by EFE, and UA caused a concentration-dependent relaxation precontracted by His (EC50 = 23.2 μg/mL). Within vivostudies, EFE strongly prolonged preconvulsive time similar to isoprenalin. The bronchodilator effects of EFE could be blocked by propranolol (1 μM),NG-nitro-l-arginine methyl ester (l-NAME) (100 μM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) (1 μM). EFE also inhibited the contraction in Ca2+-free medium and produced rightward parallel displacement of CaCl2curves. In addition, the relaxant effects of UA could only be blocked byl-NAME and ODQ. Discussion and conclusion:These results suggest that bronchodilator activities of EFE were related to activation of β-adrenoceptor and NO/cGMP pathway. Blockage of Ca2+ channels and inhibition of IP3R-mediated internal Ca2+ release were also involved. Additionally, UA produced relaxant effects by the NO/cGMP pathway. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hydrogen (H2) has been shown to have a strong antioxidant effect on preventing oxidative stress-related diseases. The goal of the present study is to determine the pharmacodynamics of H2 in a model of isoproterenol (ISO)-induced cardiac hypertrophy. Methods: Mice (C57BL/6J; 8-10 weeks of age) were randomly assigned to four groups: Control group (n = 10), ISO group (n = 12), ISO plus H2 group (n = 12), and H2 group (n = 12). Mice received H2 (1 ml/100g/day, intraperitoneal injection) for 7 days before ISO (0.5 mg/100g/day, subcutaneous injection) infusion, and then received ISO with or without H2 for another 7 days. Then, cardiac function was evaluated by echocardiography. Cardiac hypertrophy was reflected by heart weight/body weight, gross morphology of hearts, and heart sections stained with hematoxylin and eosin, and relative atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) mRNA levels. Cardiac reactive oxygen species (ROS), 3-nitrotyrosine and p67 (phox) levels were analyzed by dihydroethidium staining, immunohistochemistry and Western blotting, respectively. For in vitro study, H9c2 cardiomyocytes were pretreated with H2-rich medium for 30 min, and then treated with ISO (10 µM) for the indicated time. The medium and ISO were re-changed every 24 h. Cardiomyocyte surface areas, relative ANP and BNP mRNA levels, the expression of 3-nitrotyrosine, and the dissipation of mitochondrial membrane potential (MMP) were examined. Moreover, the expression of extracellular signal-regulated kinase1/2 (ERK1/2), p-ERK1/2, p38, p-p38, c-Jun NH2- terminal kinase (JNK), and p-JNK were measured by Western blotting both in vivo and in vitro. Results: Intraperitoneal injection of H2 prevented cardiac hypertrophy and improved cardiac function in ISO-infused mice. H2-rich medium blocked ISO-mediated cardiomyocytes hypertrophy in vitro. H2 blocked the excessive expression of NADPH oxidase and the accumulation of ROS, attenuated the decrease of MMP, and inhibited ROS-sensitive ERK1/2, p38, and JNK signaling pathways. Conclusion: H2 inhibits ISO-induced cardiac/cardiomyocytes hypertrophy both in vivo and in vitro, and improves the impaired left ventricular function. H2 exerts its protective effects partially through blocking ROS-sensitive ERK1/2, p38, and JNK signaling pathways. [ABSTRACT FROM AUTHOR]

Thestimulatory effect of the extract of Portulaca oleracea (P. olerace) on β-adrenoceptor of tracheal smooth muscle was examined. To examine β-adrenoceptor stimulatory effect, concentration response curve to isoprenaline was obtained in pre-contracted tracheal smooth muscle in the presence of three concentrations of aqueous-ethanolic extract, propranolol, and saline. Values of EC50 (the effective concentration of isoprenaline, causing 50% of maximum response) and dose ratio-1(CR-1) were measured. This effect was tested innon-incubated tracheal smooth muscle (group 1) and incubated tissues with chlorpheniramine (group 2). Concentration-response curves to isoprenaline in the presence of two higher concentrations of the extract in group 1 and all three concentrations in group 2 showed leftward shifts compared to isoprenaline curves produced in the presence of saline in both groups. EC50 obtained in the presence of propranolol was significantly higher than that of saline in both groups of experiments (p<0.05 for both cases). However, the EC50 obtained in the presence of two higher concentrations of the extract in group 1 and lower concentration in group 2 were nonsignificantly but those obtained of medium and high extract concentrations in the group 2 were significantly (p<0.05 for both cases)lower than those of saline. The values of (CR-1) obtained in the presence of all concentrations of the extract in groups1 and 2 were significantly lower than that of propranolol (p<0.05 to p<0.001).The results indicated a stimulatory effect of the P. olerace extract on ß2-adrenoceptors of tracheal smooth muscle. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca2+-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca2+-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation. [ABSTRACT FROM AUTHOR]

The objective of this work was to set up a method to formulate a complex for the assessment of myocardial infarction in vivo. Labetalol was successfully labeled with Tc using SnCl·2HO as reducing agent. Various reaction parameters were investigated to optimize the labeling yield. The labeled complex was stable for up to 24 h. The heart uptake was 35.5 % ID/Organ at 5 min post-injection, while the clearance pathways from the body behave via both the hepatobiliary and renal routes. [ABSTRACT FROM AUTHOR]

Spontaneous variation in blood pressure is defined as ‘blood pressure variability’ (BPV). Sinoaortic denervation (SAD) is characterized by BPV without sustained hypertension. In the present study, we investigated whether BPV could be related to vascular β-adrenoceptor desensitization in rats. Three days after surgery (SAD and control), aortic rings were placed in an organ chamber and the relaxation stimulated by β-adrenoceptor agonists, isoprenaline, terbutaline, BRL37344 and cyanopindolol was verified. The participation of intracellular nucleotides signaling pathways was also verified using forskolin, sodium nitroprusside and acetylcholine to induce relaxation. The effects of BPV on the increase in endothelial cytosolic Ca 2 + concentration stimulated by the β 2 -adrenoceptor agonist was examined by confocal microscopy. In addition, the vascular expression of the β 2 -adrenoceptor was also examined by immunohistochemistry. The results show that isoprenaline and terbutaline-induced relaxation was lower in the aortas of rats with BPV. Relaxation responses to other vasorelaxant compounds were similar in both groups of rats. Histological analysis revealed a lower level of β 2 -adrenoceptor and confocal microscopy showed minor cytosolic Ca 2 + concentration in endothelial cells stimulated by the β 2 -adrenoceptor agonist in rats with BPV. In conclusion, BPV leads to desensitization of the β 2 -adrenoceptor, which could contribute to worse β-adrenoceptor agonist-induced relaxation in isolated aortas. [ABSTRACT FROM AUTHOR]

β2-adrenoceptor agonists are the mainstay therapy for patients with asthma but their effectiveness in cigarette smoke (CS)-induced lung disease such as chronic obstructive pulmonary disease (COPD) is limited. In addition, bronchodilator efficacy of β2-adrenoceptor agonists is decreased during acute exacerbations of COPD (AECOPD), caused by respiratory viruses including influenza A. Therefore, the aim of the present study was to assess the effects of the β2-adrenoceptor agonist salbutamol (SALB) on small airway reactivity using mouse precision cut lung slices (PCLS) prepared from CS-exposed mice and from CS-exposed mice treated with influenza A virus (Mem71, H3N1). CS exposure alone reduced SALB potency and efficacy associated with decreased β2-adrenoceptor mRNA expression, and increased tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β) expression. This impaired relaxation was restored by day 12 in the absence of further CS exposure. In PCLS prepared after Mem71 infection alone, responses to SALB were transient and were not well maintained. CS exposure prior to Mem71 infection almost completely abolished relaxation, although β2-adrenoceptor and TNFα and IL-1β expression were unaltered. The present study has shown decreased sensitivity to SALB after CS or a combination of CS and Mem71 occurs by different mechanisms. In addition, the PCLS technique and our models of CS and influenza infection provide a novel setting for assessment of alternative bronchodilators. [ABSTRACT FROM AUTHOR]

Decreased responses of the heart to β-adrenoceptor stimulation with aging have been shown to occur merely in selected heart chambers in relation to increased catecholamine levels. However, there are no systematic studies that investigate all cardiac chambers with regard to receptor density and cAMP (adenosine 3′, 5′-cyclic monophosphate) responses. We used meat-type turkey poults (British United Turkey (B.U.T.) Big 6) with increasing age because their heart seems to decrease in weight in relation to body weight and they are often used as an animal model for heart failure. The receptor density and distribution were quantified by radioligand binding analysis using (-)-[ 125 I]-iodocyanopindolol and β-adrenoceptor subtype-specific antagonists (ICI 118.551 and CGP 20712 A) in membranes of four cardiac chambers (right and left atria and ventricles) of 6-week-, 12-week-, 16/21-week-, and 57-week-old B.U.T. BIG 6 turkeys. Receptor function was determined by measuring basal and stimulated cAMP production. In both sexes, the β-adrenoceptor density decreased significantly in all chambers with age without altered β-adrenoceptor subtype distribution. The receptor affinity (K D ) to the radioligand was similar in hearts of all age groups. β-adrenoceptor-(isoproterenol and guanosine 5′-triphosphate), G-protein-(NaF) and catalytic unit of adenylate cyclase (forskolin, Mn 2+ ) mediated cAMP responses were not chamber-dependent. Indeed, the cAMP level was significantly lower in 57-week-old hearts than in 6-week-, 12-week-, 16/21-week-old hearts. These data suggest that with increasing age and body weight, the β-adrenoceptor signal transduction pathway was highly blunted in all cardiac chambers, occurring by decreased receptor density and cAMP responses. [ABSTRACT FROM AUTHOR]

Takotsubo syndrome (TTS) is characterized by short-term contractile dysfunction with its mechanism undefined. We showed that activation of cardiac Hippo pathway mediates mitochondrial dysfunction and that stimulation of β-adrenoceptors (βAR) activates Hippo pathway. Here, we investigated the role of βAR-Hippo signaling in mediating mitochondrial dysfunction in isoproterenol (Iso)-induced TTS-like mouse model. Elderly postmenopausal female mice were administered with Iso (1.25 mg/kg/h for 23 h). Cardiac function was determined by serially echocardiography. At days 1 and 7 post-Iso exposure, mitochondrial ultrastructure and function were examined by electron microscopy and various assays. Alterations in cardiac Hippo pathway and effects of genetic inactivation of Hippo kinase (Mst1) on mitochondrial damage and dysfunction in the acute phase of TTS were investigated. Isoproterenol exposure induced acute increase in biomarkers of cardiac damage and ventricular contractile dysfunction and dilation. At day 1 post-Iso, we observed extensive abnormalities in mitochondrial ultrastructure, downregulation of mitochondrial marker proteins, and mitochondrial dysfunction evidenced by lower ATP content, increased lipid droplets, higher contents of lactate, and augmented reactive oxygen species (ROS). All changes were reversed by day 7 . βAR stimulation led to activation of cardiac Hippo pathway with enhanced expression of Hippo kinase Mst1 and inhibitory YAP phosphorylation, as well as reduced nuclear YAP-TEAD1 interaction. In mice with cardiac expression of inactive mutant Mst1 gene, acute mitochondrial damage and dysfunction were mitigated. Stimulation of cardiac βAR activates Hippo pathway that mediates mitochondrial dysfunction with energy insufficiency and enhanced ROS, promoting acute but short-term ventricular dysfunction. NEW & NOTEWORTHY Takotsubo syndrome (TTS) is featured by activation of sympatho-β-adrenoceptor (βAR) system leading to acute loss of ventricular contractile performance. However, the molecular mechanism remains undefined. We demonstrated, in an isoproterenol-induced murine TTS-like model, extensive mitochondrial damage, metabolic dysfunction, and downregulated mitochondrial marker proteins, changes temporarily associated with cardiac dysfunction. Mechanistically, stimulation of βAR activated Hippo signaling pathway and genetic inactivation of Mst1 kinase ameliorated mitochondrial damage and metabolic dysfunction at the acute phase of TTS.

GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D 2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gβγ. These results suggest that agonist-induced Gβγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H 2 S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H 2 S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca 2 + channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H 2 S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. [ABSTRACT FROM AUTHOR]

Early odor preference learning in rodents occurs within a sensitive period [≤postnatal day (P)10-12], during which pups show a heightened ability toform an odor preference when a novel odor is paired with a tactile stimulation (e.g., stroking). Norepinephrine (NE) release from the locus coeruleus during stroking mediates this learning. However, in older pups, stroking loses its ability to induce learning. The cellular and circuitry mechanisms underpinning the sensitive period for odor preference learning is not well understood. We first established the sensitive period learning model in mice - odor paired with stroking induced odor preference in P8 but not P14 mice. This learning was dependent on NE-β-adrenoceptors as it was prevented by propranolol injection prior to training. We then tested whether there are developmental changes in pyramidal cell excitability and NE responsiveness in the anterior piriform cortex (aPC) in mouse pups. Although significant differences of pyramidal cell intrinsic properties were found in two age groups (P8-11 and P14+), NE at two concentrations (0.1 and 10 μM) did not alter intrinsic properties in either group. In contrast, in P8-11 pups, NE at 0.1 μM presynaptically decreased miniature IPSC and increased miniature EPSC frequencies. These effects were reversed with a higher dose of NE (10 μM), suggesting involvement of different adrenoceptor subtypes. In P14+ pups, NE at higher doses (1 and 10 μM) acted both pre- and postsynaptically to promote inhibition. These results suggest that enhanced synaptic excitation and reduced inhibition by NE in the aPC network may underlie the sensitive period. [ABSTRACT FROM AUTHOR]

β 1 - and β 3 -adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that β 1 -AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that β 3 -AR antibodies (β 3 -ABs), produced in rats, were able to reduce cardiomyocyte contractility via β 3 -AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of G i proteins in the β 3 -ABs-induced cardiac negative inotropy, (2) to determine whether β 3 -ABs may exhibit β 3 -AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both β 1 -ABs and/or β 3 -ABs leads to the development of cardiac dysfunction in Lewis rats. Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human β 3 -AR and/or β 1 -AR. Agonistic effect of β 3 -ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, β-AR expression was quantified by immunofluorescence and RT-qPCR. SR58611A (10 nM), a preferential β 3 -AR agonist, and purified β 3 -ABs (25 μg/ml) induced a decrease in cell shortening (−39.71 ± 4.9% ( n = 10) and −17.06 ± 3.9% ( n = 10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3 μg/ml), a G i protein inhibitor ( p < 0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with β 3 -ABs ( p < 0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against β 1 -AR and both β 1 - and β 3 -AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of β 1 -ARs and an increase of β 3 -ARs in rats immunized against the β 1 -AR and an increase of both β 3 -AR and β 1 -AR in rats immunized against the β 3 -AR. For the first time, these results showed that β 3 -ABs had a β 3 -AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Objectives: Although metabolic syndrome is associated with increased sympathetic activity that chronically stimulates β-adrenoceptors, the β-adrenoceptor signaling pathway has been poorly studied in this situation. We studied the β-adrenoceptor signaling pathway in Zucker lean, obese, and obese diabetic rats. Design: Experimental, prospective study. Setting: University medical research laboratory. Subjects: Adult male Zucker lean (control), obese, and obese diabetic rats. Interventions: The effects of β-adrenoceptor stimulation were investigated in vitro in isolated left ventricular papillary muscles in control, obese, and obese diabetic rats. β1-, β2-, and β3-adrenoceptors and multidrug resistance-associated protein 4 were quantified by Western Blotting. Triglyceride, cholesterol, leptin, adiponectin, and C-peptide plasma concentrations were measured. Data are mean ± sd. Measurements and Main Results: Hyperlipidemia, high leptin, and C-peptide concentrations were observed in obese and obese diabetic strains, whereas hyperglycemia occurred only in the diabetic strain. The positive inotropic effect of isoproterenol was slightly reduced in obese rats (183% ± 11% of baseline; p = 0.003; n = 7) and markedly reduced in obese diabetic rats (137% ± 18% of baseline; p < 0.001; n = 10) when compared with control rats (210% ± 17% of baseline; n = 9). β1-adrenoceptors were down-regulated in obese (-41%; p = 0.02) and diabetic (-54%; p = 0.003) when compared with control rats, whereas β3-adrenoceptors and multidrug resistanceassociated protein expression remained unchanged. Direct stimulation of adenylate cyclase with forskolin or administration of 3',5'-cyclic adenosine monophosphate suggests that subtle impairments also occurred beside the down-regulation of β1-adrenoceptor. Conclusions: The positive inotropic effect of β-adrenoceptor stimulation is slightly decreased in Zucker obese rats and was more markedly decreased in Zucker diabetic rats. These decreases are mainly related to β1-adrenoceptor down-regulation. [ABSTRACT FROM AUTHOR]

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. β-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by β agonists has questioned the therapeutic site of action of β agonists in the bladder. The present study was done to explore the possibility that β-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing β-adrenoceptor immuno-reactivity (βAR-IR). The only structures found to express βAR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed βAR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (MR-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of β-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation. [ABSTRACT FROM AUTHOR]

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed. [ABSTRACT FROM AUTHOR]

Brain β-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of β-adrenoceptors responsible for central sympathetic outflow. In the present study, we pharmacologically examined the possibility that centrally administered isoproterenol can induce elevations of plasma noradrenaline levels in a brain prostaglandin-dependent manner. In addition, we also examined whether or not intracerebroventricular administration of isoproterenol could release endogenously synthesized prostaglandin (PG) E 2 in the hypothalamic paraventricular nucleus (PVN) by using the brain microdialysis technique combined with liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS n ). Under urethane anesthesia, a femoral venous line was inserted for infusion of saline and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed by high-performance liquid chromatography with electrochemical detection. Quantification of PGE 2 in rat PVN microdialysates was performed by the LC–ITMS n method. We demonstrated that centrally administered isoproterenol-induced elevations of plasma noradrenaline could be mediated via activation of β-adrenoceptors and the downstream phospholipase A 2 -cyclooxygenase pathway. Furthermore, PGE 2 in the PVN and the PGE 2 receptor EP 3 subtype appear to play an important role in the process. Our results suggest that central isoproterenol-induced sympathetic outflow is mediated via brain PGE 2 in a PGE 2 receptor EP 3 subtype-dependent manner. [ABSTRACT FROM AUTHOR]

4-Hydroxy-2-nonenal (4-HNE) is a major reactive aldehyde formed by lipid peroxidation, and it plays an important role in the pathogenesis of several vascular diseases, including diabetes mellitus. In this study, we examined the effects of 4-HNE on the vasodilatory mechanisms of rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of 4-HNE significantly prevented the vasodilation of retinal arterioles induced by the β-adrenoceptor agonist salbutamol but not the nitric oxide donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Iberiotoxin, an inhibitor of large-conductance K (BK) channels, significantly reduced the salbutamol-induced vasodilation of retinal arterioles. The vasodilator effect of BMS-191011, a BK channel opener, on retinal arterioles was significantly attenuated by 4-HNE. These results suggest that 4-HNE attenuates retinal vasodilator responses to β-adrenoceptor agonists through the impairment of the BK pathway. The direct effect of 4-HNE on retinal blood vessels may, therefore, contribute to the retinal vascular dysfunction observed in patients with diabetes mellitus. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to investigate whether β-adrenoceptor (β-AR) overstimulation induced by in vivo treatment with isoproterenol (ISO) alters vascular reactivity and nitric oxide (NO) production and signaling in pulmonary arteries. Main methods Vehicle or ISO (0.3 mg kg − 1 day − 1 ) was administered daily to male Wistar rats. After 7 days, the jugular vein was cannulated to assess right ventricular (RV) systolic pressure (SP) and end diastolic pressure (EDP). The extralobar pulmonary arteries were isolated to evaluate the relaxation responses, protein expression (Western blot), NO production (diaminofluorescein-2 fluorescence), and cyclic guanosine 3′,5′-monophosphate (cGMP) levels (enzyme immunoassay kit). Key findings ISO treatment induced RV hypertrophy; however, no differences in RV-SP and EDP were observed. The pulmonary arteries from the ISO-treated group showed enhanced relaxation to acetylcholine that was abolished by the NO synthase (NOS) inhibitor N ω -nitro- l -arginine methyl ester ( l -NAME); whereas relaxation elicited by sodium nitroprusside, ISO, metaproterenol, mirabegron, or KCl was not affected by ISO treatment. ISO-treated rats displayed enhanced endothelial NOS (eNOS) and vasodilator-stimulated phosphoprotein (VASP) expression in the pulmonary arteries, while phosphodiesterase-5 protein expression decreased. ISO treatment increased NO and cGMP levels and did not induce eNOS uncoupling. Significance The present data indicate that β-AR overactivation enhances the endothelium-dependent relaxation of pulmonary arteries. This effect was linked to an increase in eNOS-derived NO production, cGMP formation and VASP content and to a decrease in phosphodiesterase-5 expression. Therefore, elevated NO bioactivity through cGMP/VASP signaling could represent a protective mechanism of β-AR overactivation on pulmonary circulation. [ABSTRACT FROM AUTHOR]

Epigallocatechin-3-gallate (EGCG) is the major and most potent representative in green tea, which has been proved to modulate myocardial contractility. Whether EGCG has some negative effects on cardiac function is not known. In the present study, we investigated the effects of EGCG at different doses on cardiac contraction and explored whether β2-adrenoceptor (β2AR) was involved in EGCG-induced cardiac effects. Isolated rat hearts were mounted on the Langendorff system and perfused with different concentrations of EGCG in low or normal calcium Krebs-Henseleit (KH) buffer. The contraction of hearts was measured. Ventricular myocytes were cultured with EGCG and isoprenaline (ISO, 10−7 M) for 12 h. ICI118,551 (55 nM) was used to inhibit β2AR. Cardiomyocyte shortening, viability, and responsiveness to ISO (10−9 M) were measured. EGCG dose dependently enhanced contractility of perfused heart in low calcium KH buffer. In the normal calcium KH buffer, EGCG at low dose (20 μM) increased heart contraction, while at high dose (50 μM), it increased the incidences of arrhythmia and diastolic dysfunction. In isolated ventricular myocytes, EGCG at the concentration of 0.001 to 1.0 μΜ did not affect their contraction. However, the responsiveness to ISO and the survival of myocytes were increased by EGCG (0.01 μM). The increased responsiveness was partially abolished by ICI118,551. The data obtained in this study demonstrated that EGCG at low dose conferred cardioprotection, yet at high dose increased the incidences of arrhythmia and diastolic dysfunction. β2AR was involved in EGCG-induced cardiac effects. [ABSTRACT FROM AUTHOR]

The circulatory system is one of the first systems that develops during embryogenesis. Angiogenesis describes the formation of blood vessels as a part of the circulatory system and is essential for organ growth in embryogenesis as well as repair in adulthood. A dysregulation of vessel growth contributes to the pathogenesis of many disorders. Thus, an imbalance between pro- and antiangiogenic factors could be observed in infantile hemangioma (IH). Infantile hemangioma is the most common benign tumor during infancy, which appears during the first month of life. These vascular tumors are characterized by rapid proliferation and subsequently slower involution. Most IHs regress spontaneously, but in some cases they cause disfigurement and systemic complications, which requires immediate treatment. Recently, a therapeutic effect of propranolol on IH has been demonstrated. Hence, this nonselective β- blocker became the first-line therapy for IH. Over the last years, our understanding of the underlying mechanisms of IH has been improved and possible mechanisms of action of propranolol in IH have postulated. Previous studies revealed that gap junction proteins, the connexins (Cx), might also play a role in the pathogenesis of IH. Therefore, affecting gap junctional intercellular communication is suggested as a novel therapeutic target of propranolol in IH. In this review we summarize the current knowledge of the molecular processes leading to IH and provide new insights of how connexins might be involved in the development of these vascular tumors.

β-Adrenoceptor autoantibodies (β-AAs) affect the action potential duration (APD) in cardiomyocytes and are related to ventricular arrhythmias. The delayed rectifier potassium current ( I) plays a crucial role in APD, but the effects of β-AAs on I have not been completely illuminated. This work aimed to observe the effects of β-AAs on I and APD and further explore the mechanisms of β-AA-mediated ventricular arrhythmias. β-AAs were obtained from sera of patients with coronary heart disease (CHD) and nonsustained ventricular tachycardia. With whole-cell patch clamp technique, action potentials and I were recorded. The results illustrated 0.1 μmol/L β-AAs shortened APD at 50 % (APD) and 90 % (APD) of the repolarization. However, at 0.01 μmol/L, β-AAs had no effects on either APD or APD ( P > 0.05). At 0.001 μmol/L, β-AAs significantly prolonged APD and APD. Moreover, β-AAs (0.001, 0.01, 0.1 μmol/L) dose-dependently increased the rapidly activating delayed rectifier potassium current ( I), but similarly decreased the slowly activating delayed rectifier potassium current ( I) and increased L-type calcium currents at the different concentrations. Taken together, the IKr increase induced by high β1-AA concentrations is responsible for a significant APD reduction which would contribute to repolarization changes and trigger the malignant ventricular arrhythmias in CHD patients. [ABSTRACT FROM AUTHOR]

Takotsubo syndrome is an increasingly recognized acute cardiac affliction which is characterized by severe regional left ventricular dysfunction that cannot be explained by one or more occlusive culprit lesions of a coronary artery. A preceding somatic and/or emotional stressor can be identified in a majority of these patients and older women are overrepresented among the afflicted. Catecholamine levels are elevated in patients with takotsubo and exogenous catecholamine administration may cause or exacerbate the condition. Hence, catecholamines appear implicated in the pathogenesis. However, beyond catecholamine the pathogenesis of the takotsubo syndrome is unclear. Five distinct hypotheses have been postulated which attempt to explain why specific regions within the left ventricle are affected in takotsubo. In this manuscript we critically review these hypotheses in light of the available data. We discuss how the different hypotheses may be complementary to each other and to which extent they are contradicting one another. [ABSTRACT FROM AUTHOR]

cardiovascular physiology. Cardiomyocyte maturation occurs in late gestation in species such as humans and sheep. We tested the hypothesis that intrauterine inflammation has deleterious effects on cardiac function in preterm sheep which might be explained by altered cardiomyocyte proliferation and maturation. Pregnant ewes received an ultrasound-guided intra-amniotic injection of lipopolysaccharide (LPS) or saline 7 days prior to delivery at day 127 of pregnancy (term 147 days). Cardiac contractility was recorded in spontaneously beating hearts of the offspring, perfused in a Langendorff apparatus. Saline-filled latex balloons were inserted into the left ventricle (LV) and right ventricle (RV). Responsiveness to isoprenaline and stop-flow/reperfusion was assessed. In other experiments, hearts were perfusion-fixed, and cardiomyocyte nuclearity, volume and number were determined. ß-Adrenoceptor mRNA levels were determined in unfixed tissue. In hearts of LPS-exposed fetuses, contractility in the LV and RV was suppressed by ~40% and cardiomyocyte numbers were reduced by ~25%. Immature mono-nucleated cardiomyocytes had lower volumes (~18%), whereas mature bi-nucleated cardiomyocyte volume was~77% greater. Although basal coronary flow was significantly increased by 21± 7% in LPS-exposed hearts, following ischaemia/reperfusion (IR), end-diastolic pressure was increased 2.4± 0.3-fold and infarct area was increased 3.2± 0.6-fold compared with those in controls. Maximum responsiveness to isoprenaline was enhanced by LPS, without an increase in β-adrenoceptor mRNA, suggesting altered second messenger signalling. Intrauterine inflammation altered cardiac growth, suppressed contractile function and enhanced responsiveness to stress. Although these effects may ensure immediate survival, they probably contribute to the increased vulnerability of organ perfusion in preterm neonates. [ABSTRACT FROM AUTHOR]

Previous studies suggest that β-adrenoceptor stimulation may enhance the cardiac differentiation of mouse embryonic stem (ES) cells. It remains unclear whether the differentiations of ES cells and induced pluripotent stem (iPS) cells rely on similar molecular mechanisms. In addition, no previous studies have shown that human iPS cells express β-adrenoceptors. Therefore, in the present study, we determined the involvement of β-adrenoceptors in the differentiation of human iPS cells into mesodermal progenitor cells. The induction of differentiation of human iPS cells into kinase insert domain receptor (KDR)-positive mesodermal progenitor cells was performed on feeder cells in a differentiation medium with basic fibroblast growth factor (bFGF), bone morphogenetic protein-4 (BMP-4), and activin A. When the iPS cells that were exposed to bFGF, BMP-4, and activin A were treated with L -isoproterenol (a β-adrenoceptor agonist) for 4 days, the expression of KDR was significantly increased compared to that in the cells that were not treated with L -isoproterenol. Pretreatment of the cells with either atenolol (a β 1 -adrenoceptor antagonist) or ICI-118551 (a β 2 -adrenoceptor antagonist) significantly inhibited the L -isoproterenol-induced increase in KDR expression. In addition, pretreatment with both H89 (a protein kinase A inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly inhibited the L -isoproterenol-induced increase in KDR expression. Treatment with L -isoproterenol enhanced the phosphorylation of p38 MAPK in human iPS cells exposed to bFGF, BMP-4, and activin A. These results suggest that β-adrenoceptor stimulation in human iPS cells may enhance their differentiation into mesodermal progenitor cells via the activation of either protein kinase A or p38 MAPK. [ABSTRACT FROM AUTHOR]

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

β-Adrenoceptor antagonists are important drugs for the treatment of cardiovascular diseases and some of those drugs also block the so-called low-affinity site of β-adrenoceptors although at much higher concentrations. This low-affinity site, also identified in vivo and in human tissue, may come into play under certain pathophysiological situations including arrhythmias. The aim of our study was to determine the potency of 14 compounds chemically related to bupranolol or bevantolol and two xanthone derivatives at the low-affinity site of the β-adrenoceptor. The potency of the compounds at the low- and high-affinity site of β-adrenoceptors (β and β; both increasing heart rate) was compared in the pithed rat. One compound was also studied in the isolated rat heart and its α-adrenolytic effect determined in the isolated rat mesenteric artery. In the pithed rat, four compounds blocked the β-adrenoceptor at a ≥10-fold lower potency than the β-adrenoceptor whereas the xanthone derivative (−)-MH-3 was equipotent. In the spontaneously beating right atrium (−)-MH-3 was a non-competitive antagonist of comparable potency at either receptor; its apparent pD′ value for the β-adrenoceptor ranged from 5.6 to 6.4 under various conditions, including the Langendorff preparation. Its apparent pA at the α-adrenoceptor in the mesenteric artery was 8.4. (−)-MH-3 is the first compound with virtually the same potency at the low- and high-affinity site of β-adrenoceptors in vivo; it appears to be a non-competitive antagonist at either site in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Autoantibodies against β1-adrenoceptors (β1-ARs) have been detected in the serum of patients with various cardiac diseases; however, the pathological impact of these autoantibodies (β1-AABs) has only been evaluated in cardiac tissue. The purpose of the present study was to evaluate whether β1-AABs have deleterious effects on vascular reactivity in rats. An enzyme-linked immunosorbent assay was used to detect β1-AABs in sera from immunized rats over a period of 1–3months using the peptidic sequence of the second extracellular loop of human β1-AR. Functional studies were performed in thoracic aortic (TA) and small mesenteric artery (SMA) rings from immunized rats. Following pre-contraction with phenylephrine (0.3μM and 3μM for the TA and SMA respectively), cumulative concentration–response curves (CCRCs) to various β-AR agonists (isoproterenol, dobutamine, salbutamol, SR 58611A), acetylcholine, A23187, and sodium nitroprusside (SNP) were then plotted. The relaxations induced by dobutamine, SR 58611A, and acetylcholine were significantly impaired, but salbutamol-induced relaxations were not affected, in both vessels from immunized rats. A significant impairment of isoproterenol-induced relaxation was only observed in SMA. CCRCs to SNP were not modified in either of the vessels. A23187-induced relaxation was impaired in immunized rats. Following pretreatment with l-arginine, vasorelaxation to acetylcholine and SR 58611A was restored in immunized rats. This study demonstrates that immunization against the second extracellular loop of β1-ARs has a deleterious impact on vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signaling pathways. [Copyright &y& Elsevier]

β-Adrenoceptor agonists have recently been introduced for the symptomatic treatment of the overactive bladder syndrome. As such treatment is not curative, long-term treatment is anticipated to be required. As the susceptibility of β-adrenoceptors to undergo agonist-induced desensitization is cell type- and tissue-dependent, we have explored whether pre-treatment with a β-adrenoceptor agonist will attenuate subsequent relaxation responses to freshly added agonist using rat urinary bladder as a model. We have used the prototypical β-adrenoceptor agonist isoprenaline, the β-selective fenoterol and the β-selective CL 316,243 and mirabegron as well as the receptor-independent bladder relaxant forskolin. We show that a 6-h pre-treatment with agonist can significantly reduce subsequent relaxation against KCl-induced smooth muscle tone, but agonist-induced desensitization was also observed with longer pre-treatments or against passive tension. The agonist-induced desensitization was prominent for the β component of rat bladder relaxation but much weaker or even absent for the β component. Moreover, β-adrenoceptor agonist pre-treatment reduced contractile responses to the muscarinic agonist carbachol and the receptor-independent stimulus KCl. Taken together these data do not support the hypothesis that the long-term clinical efficacy of β-adrenoceptor agonists in the treatment of the overactive bladder syndrome will be limited by receptor desensitization. Rather they raise the possibility that such treatment may not only cause smooth muscle relaxation but also may attenuate hyper-contractility of the bladder. [ABSTRACT FROM AUTHOR]

Mirabegron is a potent and selective β-adrenoceptor agonist developed for the treatment of overactive bladder. In vitro studies demonstrated that mirabegron partly acts as a (quasi-) irreversible, metabolism-dependent inhibitor of CYP2D6. The effect of steady-state mirabegron on single doses of the sensitive CYP2D6 substrates metoprolol (100 mg) and desipramine (50 mg) was assessed in two open-label, one-sequence crossover studies in healthy subjects (CYP2D6 extensive metabolizers). Mirabegron 160 mg/day increased metoprolol maximum plasma concentration ( C) 1.90-fold (90 % confidence interval [CI] 1.54; 2.33) and total exposure (AUC) 3.29-fold (90 % CI 2.70; 4.00) in 12 males (study 1). Mean metoprolol half-life increased from 2.96 to 4.11 h. α-Hydroxymetoprolol C and AUC to last measurable concentration decreased 2.6-fold and 2.2-fold, respectively. In study 2, mirabegron 100 mg/day increased desipramine C 1.79-fold (90 % CI 1.69; 1.90) and AUC 3.41-fold (90 % CI 3.07; 3.80) in 14 males and 14 females. Mean desipramine half-life increased from 19.5 to 35.8 h. C of 2-hydroxydesipramine decreased ~twofold, while AUC increased ~1.3-fold. Desipramine was administered again 2 weeks after the last mirabegron dose. Desipramine C and AUC were still ~1.13-fold increased; the 90 % CIs fell within the 0.80-1.25 interval. All treatments were well tolerated. In conclusion, mirabegron is a moderate CYP2D6 inhibitor (ratio and 90 % CI <5.0). [ABSTRACT FROM AUTHOR]