Activation Of Β3-adrenoceptors Research Articles

Beta 3-adrenoceptor agonism ameliorates early-life stress-induced visceral hypersensitivity in male rats.

Visceral hypersensitivity, a hallmark of disorders of the gut-brain axis, is associated with exposure to early-life stress (ELS). Activation of neuronal β3-adrenoceptors (AR) has been shown to alter central and peripheral levels of tryptophan and reduce visceral hypersensitivity. In this study, we aimed to determine the potential of a β3-AR agonist in reducing ELS-induced visceral hypersensitivity and possible underlying mechanisms. Here, ELS was induced using the maternal separation (MS) model, where Sprague Dawley rat pups were separated from their mother in early life (postnatal day 2-12). Visceral hypersensitivity was confirmed in adult offspring using colorectal distension (CRD). CL-316243, a β3-AR agonist, was administered to determine anti-nociceptive effects against CRD. Distension-induced enteric neuronal activation as well as colonic secretomotor function were assessed. Tryptophan metabolism was determined both centrally and peripherally. For the first time, we showed that CL-316243 significantly ameliorated MS-induced visceral hypersensitivity. Furthermore, MS altered plasma tryptophan metabolism and colonic adrenergic tone, while CL-316243 reduced both central and peripheral levels of tryptophan and affected secretomotor activity in the presence of tetrodotoxin. This study supports the beneficial role of CL-316243 in reducing ELS-induced visceral hypersensitivity, and suggests that targeting the β3-AR can significantly influence gut-brain axis activity through modulation of enteric neuronal activation, tryptophan metabolism, and colonic secretomotor activity which may synergistically contribute to offsetting the effects of ELS.

Concentration-dependent alpha1-Adrenoceptor Antagonism and Inhibition of Neurogenic Smooth Muscle Contraction by Mirabegron in the Human Prostate

Introduction: Mirabegron is available for treatment of storage symptoms in overactive bladder, which may be improved by β3-adrenoceptor-induced bladder smooth muscle relaxation. In addition to storage symptoms, lower urinary tract symptoms in men include obstructive symptoms attributed to benign prostatic hyperplasia, caused by increased prostate smooth muscle tone and prostate enlargement. In contrast to the bladder and storage symptoms, effects of mirabegron on prostate smooth muscle contraction and obstructive symptoms are poorly understood. Evidence from non-human smooth muscle suggested antagonism of α1-adrenoceptors as an important off-target effect of mirabegron. As α1-adrenergic contraction is crucial in pathophysiology and medical treatment of obstructive symptoms, we here examined effects of mirabegron on contractions of human prostate tissues and on proliferation of prostate stromal cells. Methods: Contractions were induced in an organ bath. Effects of mirabegron on proliferation, viability, and cAMP levels in cultured stromal cells were examined by EdU assays, CCK-8 assays and enzyme-linked immunosorbent assay. Results: Mirabegron in concentrations of 5 and 10 μM, but not 1 µM inhibited electric field stimulation-induced contractions of human prostate tissues. Mirabegron in concentrations of 5 and 10 µM shifted concentration response curves for noradrenaline-, methoxamine- and phenylephrine-induced contractions to the right, including recovery of contractions at high concentrations of α1-adrenergic agonists, increased EC50 values, but unchanged Emax values. Rightshifts of noradrenaline concentration response curves and inhibition of EFS-induced contractions were resistant to L-748,337, l-NAME, and BPIPP. 1 µM mirabegron was without effect on α1-adrenergic contractions. Endothelin-1- and U46619-induced contractions were not affected or only inhibited to neglectable extent. Effects of mirabegron (0.5–10 µM) on proliferation and viability of stromal cells were neglectable or small, reaching maximum decreases of 8% in proliferation assays and 17% in viability assays. Mirabegron did not induce detectable increases of cAMP levels in cultured stromal cells. Conclusion: Mirabegron inhibits neurogenic and α1-adrenergic human prostate smooth muscle contractions. This inhibition may be based on antagonism of α1-adrenoceptors by mirabegron, and does not include activation of β3-adrenoceptors and requires concentrations ranging 50-100fold higher than plasma concentrations reported from normal dosing. Non-adrenergic contractions and proliferation of prostate stromal cells are not inhibited by mirabegron.

Role of β3-Adrenoceptor Activation in Changes of Pulmonary Microhemodynamics after Experimental Pulmonary Thromboembolism.

Changes in pulmonary microhemodynamics during modelling of pulmonary thromboembolism against the background of nebivolol and mirabegron pretreatment were studied in isolated perfused rabbit lungs. In both cases, the pulmonary artery pressure and precapillary and pulmonary vascular resistance increased to a greater extent than in control animals, but the increase in capillary hydrostatic pressure was less pronounced. The postcapillary resistance did not change in pulmonary embolism against the background of nebivolol administration and increased in case of mirabegron pretreatment; capillary filtration coefficient after nebivolol pretreatment increased less markedly than after mirabegron administration. The increase in capillary filtration coefficient after activation of β3-adrenoceptors with the specified drugs depended on the ratio of constriction of pulmonary veins, capillary hydrostatic pressure, and endothelial permeability.

Role of Sympathetic Nerves and Adipocyte Catecholamine Uptake in the Vasorelaxant Function of Perivascular Adipose Tissue.

Healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect on resistance arteries which is vital in regulating arterial tone. Activation of β3-adrenoceptors by sympathetic nerve-derived NA (noradrenaline) may be implicated in this effect and may stimulate the release of the vasodilator adiponectin from adipocytes. Understanding the mechanisms responsible is vital for determining how PVAT may modify vascular resistance in vivo. Electrical field stimulation profiles of healthy C57BL/6J mouse mesenteric resistance arteries were characterized using wire myography. During electrical field stimulation, PVAT elicits a reproducible anticontractile effect, which is endothelium independent. To demonstrate the release of an anticontractile factor, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT-denuded vessel. Post-transfer contractility was significantly reduced confirming that stimulated PVAT releases a transferable anticontractile factor. Sympathetic denervation of PVAT using tetrodotoxin or 6-hydroxydopamine completely abolished the anticontractile effect. β3-adrenoceptor antagonist SR59203A reduced the anticontractile effect, although the PVAT remained overall anticontractile. When the antagonist was used in combination with an OCT3 (organic cation transporter 3) inhibitor, corticosterone, the anticontractile effect was completely abolished. Application of an adiponectin receptor-1 blocking peptide significantly reduced the anticontractile effect in +PVAT arteries. When used in combination with the β3-adrenoceptor antagonist, there was no further reduction. In adiponectin knockout mice, the anticontractile effect is absent. The roles of PVAT are 2-fold. First, sympathetic stimulation in PVAT triggers the release of adiponectin via β3-adrenoceptor activation. Second, PVAT acts as a reservoir for NA, preventing it from reaching the vessel and causing contraction.

Inhibition of cholinergic neurotransmission by β3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders

The direct detrusor relaxant effect of β3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by β3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking β3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by β3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.

Eosinophils are key regulators of perivascular adipose tissue and vascular functionality

Obesity impairs the relaxant capacity of adipose tissue surrounding the vasculature (PVAT) and has been implicated in resultant obesity-related hypertension and impaired glucose intolerance. Resident immune cells are thought to regulate adipocyte activity. We investigated the role of eosinophils in mediating normal PVAT function. Healthy PVAT elicits an anti-contractile effect, which was lost in mice deficient in eosinophils, mimicking the obese phenotype, and was restored upon eosinophil reconstitution. Ex vivo studies demonstrated that the loss of PVAT function was due to reduced bioavailability of adiponectin and adipocyte-derived nitric oxide, which was restored after eosinophil reconstitution. Mechanistic studies demonstrated that adiponectin and nitric oxide are released after activation of adipocyte-expressed β3 adrenoceptors by catecholamines, and identified eosinophils as a novel source of these mediators. We conclude that adipose tissue eosinophils play a key role in the regulation of normal PVAT anti-contractile function.

Repeated Cold Stress Enhances the Acute Restraint Stress-Induced Hyperthermia in Mice.

The rodents exposed to repeated cold stress according to a specific schedule, known as specific alternation of rhythm in temperature (SART), exhibit autonomic imbalance, and is now used as an experimental model of fibromyalgia. To explore the susceptibility of SART-stressed animals to novel acute stress, we tested whether exposure of mice to SART stress for 1 week alters the extent of acute restraint stress-induced hyperthermia. Mice were subjected to 7-d SART stress sessions; i.e., the mice were alternately exposed to 24 and 4°C at 1-h intervals during the daytime (09:00-16:00) and kept at 4°C overnight (16:00-09:00). SART-stressed and unstressed mice were exposed to acute restraint stress for 20-60 min, during which rectal temperature was monitored. Serum corticosterone levels were measured before and after 60-min exposure to restraint stress. SART stress itself did not alter the body temperature or serum corticosterone levels in mice. Acute restraint stress increased the body temperature and serum corticosterone levels, both responses being greater in SART-stressed mice than unstressed mice. The enhanced hyperthermic responses to acute restraint stress in SART-stressed mice were significantly attenuated by SR59230A, a β3 adrenoceptor antagonist, but unaffected by diazepam, an anxiolytic, mifepristone, a glucocorticoid receptor antagonist, or indomethacin, a cyclooxygenase inhibitor. These results suggest that SART stress enhances the susceptibility of mice to acute restraint stress, characterized by increased hyperthermia and corticosterone secretion, and that the increased hyperthermic responses to acute stress might involve accelerated activation of sympathetic β3 adrenoceptors, known to regulate non-shivering thermogenesis in the brown adipose tissue.

YIA 03-09 NORADRENERGIC RECEPTOR FUNCTION IN HEALTHY AND OBESE PERIVASCULAR ADIPOSE TISSUE

Objective: Perivascular adipose tissue (PVAT) exerts an anti-contractile effect vital to regulating blood pressure. Evidence suggests that the sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of β3-adrenoceptors. Therefore it was decided to examine β3-adrenoceptor function in obesity. Design and Method: Electrical field stimulation (EFS) of healthy and obese mouse mesenteric arteries (<200 μm, +/-PVAT) were characterised using wire myography (0.1–30 Hz, 20V, 0.2ms pulse duration, 4s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. β3-adrenoceptor function was investigated using the agonist CL-316,243 (10 μM) and antagonist SR59203A (100nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100 μM), and NOS activator histamine (100 μM). Results: Healthy PVAT elicits an anti-contractile effect which was lost in obesity. Inhibition of β3-adrenoceptors significantly reduced the anti-contractile effect, whereas activation of β3-adrenoceptors in obese PVAT using CL-316,243 did not restore function. Solution transfer from stimulated healthy exogenous PVAT to a –PVAT vessel significantly reduced contraction, confirming the release of a transferable factor. The release of this factor could be inhibited using SR59230A. Solution transfer from obese PVAT had no effect on contraction, and again could not be restored using CL-316,243. In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect. In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function. Conclusions: These results demonstrate that healthy PVAT releases an anti-contractile factor via activation of β3-adrenoreceptors, which downstream triggers the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by β3-activation, but is restored by activation of NOS. This suggests that in obesity β3-adrenoreceptors must be downregulated or desensitised, leading to a loss of anti-contractile function.

Modulation of nerve-evoked contractions by β3-adrenoceptor agonism in human and rat isolated urinary bladder

Activation of β3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of β3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of β3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of β3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αβ-methylene adenosine triphosphate (αβ-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the β3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5–1μM) were inhibited 25% by isoproterenol (3μM) while contractions to 10Hz in the same strip were inhibited 67%. The selective β3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αβ-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of β3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by β3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional β3-adrenoceptors may inhibit neurotransmitter release.

Functional BK channels facilitate the β3-adrenoceptor agonist-mediated relaxation of nerve-evoked contractions in rat urinary bladder smooth muscle isolated strips

The large-conductance voltage- and Ca2+-activated K+ (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility thus facilitating urinary bladder function. Recent findings suggest that activation of β3-adrenoceptors causes DSM relaxation. However, it is unknown whether the β3-adrenoceptor-mediated DSM relaxation is BK channel-dependent during nerve-evoked contractions. To test this hypothesis, we induced nerve-evoked contractions in rat DSM isolated strips by using a tissue bath system equipped with platinum electrodes for electrical field stimulation (EFS). (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL37344), a β3-adrenoceptor agonist, significantly decreased the amplitude and muscle force of the 20Hz EFS-induced DSM contractions in a concentration-dependent manner. The BRL37344 inhibitory effect was significantly antagonized by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A), a β3-adrenoceptor antagonist. We further isolated the cholinergic from the purinergic component of the 0.5–50Hz EFS-induced DSM contractions by using selective inhibitors, atropine as well as suramin and α,β-methylene-ATP. We found that BRL37344 inhibited both the purinergic and cholinergic components of the nerve-evoked contractions in rat DSM isolated strips. The pharmacological blockade of the BK channels with iberiotoxin, a selective BK channel inhibitor, increased the amplitude and muscle force of the 20Hz EFS-induced contractions in rat DSM isolated strips. In the presence of iberiotoxin, there was a significant reduction in the BRL37344-induced inhibition of the 20Hz EFS-induced contractions in rat DSM isolated strips. These latter findings suggest that BK channels play a critical role in the β3-adrenoceptor-mediated inhibition of rat DSM nerve-evoked contractions.

Selective Activation of β3 Adrenoceptors Protects Against Type 2 Diabetes-induced eNOS Uncoupling, Oxidative Na+–K+ Pump Inhibition and Vascular Dysfunction

Conclusion: In a private hospital setting, percutaneous renal denervation is safe and efficacious in reducing blood pressure in patients with resistant hypertension. http://dx.doi.org/10.1016/j.hlc.2013.05.060

Activation of β3-Adrenoceptors Induces Increase in Intracellular Free Zinc Ion via No Signaling Pathway in Hyperglycemic Cardiomyocytes

Hyperglycemia is a major etiological factor causing cardiovascular dysfunction. Changes in intracellular ionic homeostasis via a series of biochemical changes initiated by hyperglycemia directly affect cellular function resulting abnormal cardiac remodeling and development of diabetic complications. Since hyperglycemia regulates free cytosolic Zn2+ ([Zn2+]i) and attenuates protein S-nitrosylation via NO-cGMP-PKG, which induces depressed cardiac contractility, in part contribution of activated β3-adrenergic receptor (β3-AR). We postulated a possible cross-talk between hyperglycemia, activation of β3-AR and NO availability, [Zn2+]i increase and contractile dysfunction. [Zn2+]i levels were determined by imaging freshly isolated adult rat cardiomyocytes from either normoglycemic or hyperglycemic rat loaded with FluoZin-3. We investigated the distribution of β-ARs subtypes by measuring the expression/protein levels of β1-, β2-, β3-AR as well as their left ventricular agonist responsiveness using isolated perfused hearts from diabetic rats. We found that hyperglycemia influenced both function and the mRNA/protein levels of selective β1- and β3-AR although the total β-AR did not change. In vitro experiments showed that β3-AR agonist (BRL-37,344) induced significant increase in [Zn2+]i while this effect was prevented with L-NAME exposure in normoglycemic cardiomyocytes. Our data demonstrated that either chronic or acute hyperglycemia initiates a series of interconnected biochemical changes in cardiomyocytes including a cross-talk between activation of β3-AR and NO signaling pathway including increased [Zn2+]i and then this cross-talk meadiates abnormal cardiac remodeling and the development of diabetic cardiomyopathy. Therefore, it can be proposed a pathway that a dynamic association of intracellular free Zn2+ with sulfur in protein cysteine clusters under hyperglycemia, from which the metal is released by NO and other thiol oxidant species is one of the mechanisms which plays important role in the Zn2+ contribution to maintaining cellular redox balance. (Supported by TUBITAK-SBAG-109S267 & 111S042)

Comparison of isoproterenol with BRL37344 in activation of β3-adrenoceptors to inhibit the uptake of [ 14C]deoxy- d-glucose and translocation of glucose transporter (GLUT4) to membrane fraction in rat adipocytes

In an attempt to understand the role of β 3-adrenoceptors in the regulation of glucose uptake, the effect of isoproterenol was compared with BRL37344 in isolated white adipocytes of the rat using [ 14C]deoxy- dD-glucose as an indicator. In the presence of BRL37344, the specific agonist of β 3-adrenoceptors, spontaneous uptake of [ 14C]deoxy- d-glucose (glucose uptake) into adipocytes was markedly attenuated. Similar concentration-dependent inhibition of glucose uptake was also observed in the samples treated with isoproterenol, an agonist for all kinds of β-adrenoceptors. Action of BRL37344 was blocked by propranolol at concentrations sufficient to abolish the activity of isoproterenol. Pindolol reversed BRL37344-induced inhibition more effectively than propranolol. Moreover, unlike the action of isoproterenol, BRL37344 did not modify the insulin-stimulated glucose uptake. Translocation of glucose transporter (GLUT4) from cytosol to membrane stimulated with insulin was reduced by isoproterenol but not by BRL37344. Combination with the findings that isoproterenol prolonged the time for insulin to reach maximum stimulation of glucose uptake, leads to the conclusion that delay of insulin action by isoproterenol can be considered as one of the mechanisms for this inhibition. The results obtained suggest that BRL37344 decreased the spontaneous uptake of glucose via an activation of β 3-adrenoceptors while the insulin stimulated glucose uptake was inhibited by isoproterenol but not by BRL37344.