beta-AR Antagonist Research Articles

Beta adrenergic receptor antagonist can modify Pseudomonas aeruginosa biofilm formation in vitro: Implications for chronic wounds.

Non-healing wounds are a major medical challenge, affecting over 6.5million people in the US alone, with associated healthcare costs of about $16 billion annually. They can result in prolonged hospitalizations, work loss, disability, poor quality of life, and in diabetic patients with foot ulcers, amputation of the affected limb in 25% of patients. Though chronic ulcers may arise from different underlying diseases, the unifying feature is chronic infection, driving persistent inflammation that prolongs the healing process. One of the most frequently cultured or genetically identified pathogens in skin wounds is Pseudomonas aeruginosa. This species avidly forms biofilms in the wound that impede bacterial eradication by the host's immune mechanisms and limit efficacy of systemic antibiotics. Thus, non-antibiotic approaches to limit the growth and biofilm formation of this wound pathogen would be an advance in the treatment of chronic wounds. Prior work has demonstrated that the growth of other microbial species can be modulated by catecholamine agonists and antagonists of the adrenergic receptors (ARs). Here, we demonstrate that not only can the growth of this common wound pathogen be modulated by catecholamines, but also that the beta-AR antagonists can significantly decrease their growth, and importantly, limit their ability to form biofilms. These findings suggest that beta adrenergic antagonists may have a therapeutic role in the treatment of chronic skin wounds.

Abstract 9032: Restoration of Normal Cardiomyocyte Basal and β-Adrenergic Receptor (AR) Subtype Modulation in Diabetic Cardiomyopathy by Chronic Selective Beta3-AR Antagonist in a Mouse Model of Type 2 Diabetes

Background: We have shown that L-748,337 (L-7), a selective β 3 -adrenergic receptor (AR) antagonist reversed LV dysfunction and β-AR desensitization in diabetes (DM) caused diabetic cardiomyopathy (DCM). The mechanism is unclear. Recent evidence suggests that normal myocardial performance depends on the balance in myocyte β 3 -, β 1 -, and β 2 -AR. Pivotal restructuring of β-AR system resulting in the decline of β-AR reserve plays a crucial role in DCM. We assessed the hypothesis that chronic L-7 would prevent DCM-induced abnormalities of β-AR subtype-stimulated regulation on intrinsic LV myocyte function and [Ca 2+ ] i regulation, thus restoring cardiac function. Methods: Studies were conducted in 3 groups of female mice: Type 2 DM (T2) (n=9), induced with high-fat diet (HFD) intake for 14 weeks (W), but after fed HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. for 5 days); T2/L-7 (n=7), T2 mice at 10 W received L-7 (10 -7 M/kg/day, mini-pump) for 4 W; and Controls (C)(n=9). We compared LV myocyte contractile and [Ca 2+ ] iT responses to β-AR subtype stimulation by random exposure of myocytes to Isoproterenol (ISO,10 -8 M) or a selective β 1 -, β 2 -, or β 3 -agonist, Norepinephrine (NE, 10 -7 M), Zinterol (ZIN, 10 -5 M) and BRL-37,344 (BRL, 10 -8 M), respectively, during drug perfusion. Results: T2 and T2/L-7 mice had mean blood glucose levels of 388 mg/dl and 369 mg/dl, respectively. Versus C, only in T2, basal LV myocyte contractility (dL/dt max , 76 vs 133 μm/s), relaxation and [Ca 2+ ] iT (0.16 vs 0.21) were significantly decreased. ISO-stimulated dL/dt max (40% vs 58%) was attenuated accompanied by a diminished NE-mediated increase in dL/dt max (28% vs 44%), but enhanced BRL-induced decreases in dL/dt max (29% vs 12%).The response of dL/dt max (24% vs 15%) to ZIN was increased. Importantly, in T2/L-7 myocytes, the basal dL/dt max (128 μm/s) and [Ca 2+ ] iT (0.21) remained close to control values with preserved β-stimulated positive modulation on cell contraction. The increases in dL/dt max in response to ISO (57%), NE (40%) or ZIN (15%) were similar to that in control myocytes, respectively. Conclusions: Chronic β 3 -AR antagonist reverses β-AR signaling defects, resensitizing the β-AR subtype system modulation on LV myocytes function, thus playing a salutary role in DCM.

Abstract # 3159 Cytomegalovirus amplifies CD8 + T cell mobilization to psychological stress in humans, via a beta-adrenergic dependent mechanism linked to T-bet/Th1 effector cell differentiation

Psychological stress induces a beta-adrenergic dependent mobilization of CD8 + T lymphocytes (CTL) into the peripheral blood. Here we identified infection with Cytomegalovirus (CMV) as a major determinant of this mobilization, and characterized the cellular and transcriptional alterations that underlie enhanced responsivity. During acute stress (TSST), CMV-positive individuals (N = 35) demonstrated a 3-fold greater CTL mobilization compared to CMV-negative individuals (N = 48). Enhanced CTL mobilization was entirely confined to cells with an effector-memory phenotype (EMRA), which become markedly enriched in CMV-infected individuals. A beta-adrenergic receptor (beta-AR) dependent mechanism was confirmed by studies showing that stress-induced EMRA mobilization; (1) correlated with catecholamine release; (2) was completely abrogated by the beta-AR antagonist propranolol, and; (3) was reproduced by infusion of the beta-AR agonist isoproterenol. Micro-array analyses demonstrated selective 8-fold up-regulation of the beta2-AR gene (ADRB2) in EMRA as well as CMV-specific CTL. Longitudinal analyses confirmed that CMV-infection de novo induced rapid and lasting ADRB2 up-regulation in CTL, in conjunction with a Th1 T-bet/EOMES transcriptional profile. Subsequent studies in vitro replicated that functional beta2-AR expression increased with progressive CTL differentiation assessed as T-bet/Th1 expression. The present study identified a novel mechanism through which host–microbe interactions can regulate immune system responses to stress: CMV-infection induced T-bet differentiation accompanied by beta2-AR upregulation, which hereby enriched the CTL compartment with EMRA cells exhibiting an increased adrenergic sensitivity and enhanced mobilization during stress.

Abstract 1256: The chemopreventive effects of carvedilol on skin carcinogenesis

Abstract Skin cancer, the majority of which is non-melanoma skin cancer (NMSC), is the most common type of cancer in the US. The rate of NMSC is increasing mainly due to depletion of the ozone layer and increased exposure to ultraviolet radiation. To reduce the risk of skin cancer, it is recommended to all individuals to limit sun exposure and use sunscreens. Despite these efforts, the incidence of NMSC continues to rise. Novel chemopreventive targets and nontoxic agents are needed. Epidemiological studies identified an association of psychosocial factors such as chronic stress or depression with cancer onset and progression. These factors are partly mediated by activation of the sympathetic nervous system which results in release of norepinephrine and epinephrine, the effects of which are mediated through the alpha- and beta-adrenergic receptors (alpha- and beta-AR). Further, the use of beta-AR antagonists (beta-blockers) has been associated with reduced cancer incidence. However, whether beta-blockers have chemopreventive activity or whether beta-ARs contribute to carcinogenesis is unknown. To determine whether agonizing or antagonizing beta-ARs affect skin cell transformation, we tested the beta-AR agonist isoproterenol (Iso) and antagonist carvedilol on epidermal growth factor (EGF)-mediated transformation of JB6 P+ cells (a skin cell model to study tumor promotion). The cells were treated with EGF (10 ng/mL) plus 0.1 µM or 1.0 µM Iso, yet no effect was observed. However, treatment with carvedilol dose-dependently inhibited the formation of EGF-induced soft agar colonies. Such effect was not caused by growth inhibition, because cytotoxicity and inhibition of cell proliferation were not observed for tested concentrations. Next, the expression of beta1-, 2- and 3-AR was determined in JB6 cells using RT-PCR. The results showed that only beta2-AR was detectable. We also determined whether beta-ARs are expressed in other types of cancer. In tissues derived from a rat model of 7, 12-dimethylbenz[α]anthracene (DMBA)-induced mammary cancer, expression of beta1- and 2-AR was up-regulated in tumors in comparison with normal tissues. The human cancer cell lines A549 and MDA-MB-231 also express beta2-AR. To determine the in vivo chemopreventive activity of carvedilol, we used a skin hyperplasia model in SENCAR mice induced by topical treatment with 100 nM DMBA twice a week for four weeks. Carvedilol was tested topically (5 and 10 µM) or orally (5 and 20 mg/kg), beginning at two weeks before the first dose of DMBA, three times a week, for six weeks. DMBA alone increased the epidermal thickness from the average of 77.8 + 13.6 nm in normal skin to 294 + 49.7 nm in DMBA-treated skin. Both topical and oral carvedilol treatment inhibited DMBA-induced hyperplasia (P < 0.05). In conclusion, these results suggest that beta-blockers may have chemopreventive activity. This study also suggests that beta-ARs may serve as a novel target for cancer prevention. Citation Format: Andy Chang, Mandy Liu, Steven Yeung, Jijun Hao, Cyrus Parsa, Robert Orlando, Bradley Andresen, Ying Huang. The chemopreventive effects of carvedilol on skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2014-1256

Beta-Adrenoreceptor antagonists reduce cancer cell proliferation, invasion, and migration

Context: Propranolol, atenolol, and ICI118,551 are non-selective β-adrenergic receptor (AR), β1-AR, and β2-AR antagonists, respectively.Objective: We investigated the efficacy of propranolol, atenolol, and ICI118,551 on proliferation, migration, and invasion of non-stimulated breast (MCF7), colon (HT-29), and hepatocellular (HepG2) cancer cells.Materials and methods: β-AR expression profiling of cells was performed by real time PCR. Cell proliferation was determined by MTT. Boyden chamber and scratch assays were performed to evaluate invasion and migration.Results and discussion: All cell lines expressed β-ARs. ICI118,551 was the most cytotoxic, whereas atenolol was the least effective β-AR antagonist for 24, 48, and 72 h. Cell invasion was inhibited by ICI118,551 (45, 46, and 50% for MCF7, HT29, and HepG2, respectively) and propranolol (72, 65, and 90% for MCF7, HT29, and HepG2, respectively). Propranolol, atenolol, and ICI118,551 reduced migration of MCF7, HT-29, and HepG2 cells to varying extents depending on the application concentration and duration. Propranolol and atenolol reduced migration of MCF7 and HT-29 in a concentration-dependent manner, whereas migration of these cells decreased after 48 and 72 h of ICI118,551 applications.Conclusion: Beta2-AR antagonist seemed to be the most cytotoxic β-blocker on non-stimulated cancer cells. Propranolol and ICI118,551 were more effective than atenolol in inhibiting invasion and migration of non-stimulated MCF7 and HT-29 cells; ICI118,551 being the most potent. Concordantly, β2-selective blockage seemed to be more effective for non-stimulated cells. Effect of the selective β-AR antagonists showed variation depending on the concentration, incubation time, and histological origin of cells.

Attenuation of beta2-adrenergic receptors and homocysteine metabolic enzymes cause diabetic cardiomyopathy

Although adrenergic receptors (AR) and hyperhomocysteinemia (HHcy) are implicated in heart failure, their role in diabetic cardiomyopathy is not completely understood. We tested the hypothesis that glucose mediated depletion of beta2-AR and HHcy impair contractile function of cardiomyocytes leading to diabetic cardiomyopathy. To prove the hypothesis, cardiac function was assessed in 12week male diabetic Ins2+/− Akita and C57BL/6J mice by echocardiography, pressure–volume loop, and contractile function of cardiomyocytes. The results revealed cardiac dysfunction in Akita. To investigate the mechanism, the levels of beta2-AR, GLUT4, sarcoplasmic reticulum calcium ATP-ase-isoform 2 (SERCA-2) and homocysteine (Hcy) metabolic enzymes-cystathionine beta synthase (CBS), cystathionine gamma lyase (CTH), and methyl tetrahydrofolate reductase (MTHFR) were determined in the heart. It revealed down-regulation of beta2-AR, GLUT4, SERCA-2, CBS, CTH, and MTHFR in Akita. Attenuation of beta2-AR in hyperglycemic condition was also confirmed in cardiomyocytes at in vitro level. Interestingly, the ex vivo treatment of cardiomyocytes with beta2-AR antagonist deteriorated whereas beta-AR agonist ameliorated contractile function. It points to the involvement of beta2-AR in diabetic cardiomyopathy. We conclude that degradation of beta2-AR and impairment of Hcy metabolism is implicated in diabetic cardiomyopathy.

Modulation of L-type Ca2+ channels by beta3-adrenoceptor activation and the involvement of nitric oxide.

Both beta1- and beta2-adrenoceptors (AR) in cardiac tissues are responsible for the excitatory effect of catecholamines. Recent evidence demonstrated the presence of another subtype of beta-AR (beta3-AR) in cardiac ventricular preparation. Activation of beta3-AR elicited a depressant response on ventricular contraction. The underlying mechanism(s) for the negative inotropism is relatively unknown. We investigated the effects of beta3-AR activation on basal voltage-dependent Ca2+ channel (I(CaL)) amplitude of the guinea pig enzyme-dissociated single ventricular myocytes using amphotericin B (200 microg/mL) perforated-patch whole-cell patch-clamp techniques (approximately 22 degrees C). Application of (-)-isoprenaline ((-)-ISO) (100 nM, a nonselective beta-AR agonist) increased the basal I(CaL) amplitude (approximately 210% of control) (n = 8). However, in the presence of nadolol (1 degreesM, a beta1-/beta2-AR antagonist), the stimulatory effect of (-)-ISO on I(CaL )was abolished and a slowly developed inhibition of the basal I(CaL) was recorded (approximately 80% of control) (n = 9). A smaller degree of inhibition was observed with BRL 37344 (100 nM, a selective beta3-AR agonist) (58% of control) (n = 7). The inhibitory effect of (-)-ISO (with nadolol) and BRL 37344 persisted after washout. Pretreating the ventricular myocytes with L-NAME (0.3 microM, a nitric oxide synthase inhibitor), but not D-NAME (0.3 microM), abolished the inhibitory effect of (-)-ISO and BRL 37344 (n = 7-9). The results suggest that beta3-ARs are present in ventricular myocytes. Activation of the beta3-AR resulted in an inhibition of the basal I(CaL) amplitude probably due to the formation of nitric oxide.

Preclinical evaluation of an 18F-labelled β1-adrenoceptor selective radioligand based on ICI 89,406

Radioligand binding studies indicate a down-regulation of myocardial beta(1)-adrenoceptors (beta(1)-AR) in cardiac disease which may or may not be associated with a decrease in beta(2)-ARs. We have chosen ICI 89,406, a beta(1)-selective AR antagonist, as the lead structure to develop new beta(1)-AR radioligands for PET and have synthesised a fluoro-ethoxy derivative (F-ICI). (S)-N-[2-[3-(2-Cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N'-[4-(2-[(18)F]fluoro-ethoxy)-phenyl]-urea ((S)-[(18)F]F-ICI) was synthesised. Myocardial uptake of radioactivity after intravenous injection of (S)-[(18)F]F-ICI into adult CD(1) mice or Wistar rats was assessed with positron emission tomography (PET) and postmortem dissection. Metabolism was assessed by high-performance liquid chromatography analysis of plasma and urine. The heart was visualised with PET after injection of (S)-[(18)F]F-ICI but neither unlabelled F-ICI nor propranolol (non-selective beta-AR antagonist) injected 15 min after (S)-[(18)F]F-ICI affected myocardial radioactivity. Ex vivo dissection demonstrated that predosing with propranolol or CGP 20712 (beta(1)-selective AR-antagonist) did not affect myocardial radioactivity. Radiometabolites rapidly appeared in plasma and both (S)-[(18)F]F-ICI and radiometabolites accumulated in urine. Myocardial uptake of (S)-[(18)F]F-ICI after intravenous injection was mainly at sites unrelated to beta(1)-ARs. (S)-[(18)F]F-ICI is not a suitable beta(1)-selective-AR radioligand for PET.

HIF-1α links β-adrenoceptor agonists and pancreatic cancer cells under normoxic condition

To examine whether beta-adrenoceptor (beta-AR) agonists can induce hypoxia-inducible factor (HIF)-1alpha accumulation which then up-regulate the expression of its target genes in pancreatic cancer cells at normoxia, and to further elucidate the mechanism involved. Pulse-chase assay, RT-PCR, and Western blot were employed to detect the effects of beta-AR agonists and antagonists, siRNA as well as several inhibitors of signal transduction pathways on MIA PaCa2 and BxPC-3 pancreatic cancer cells. Treatment of pancreatic cancer cell lines with beta-AR agonists led to accumulation of HIF-1alpha and then up-regulated expression of its target genes independently of oxygen levels. The induction was partly or completely inhibited not only by beta-AR antagonists but also by inhibitors of PKA transduction pathways and by siHIF-1alpha. Both beta1-AR and beta2-AR agonists produced the above-mentioned effects, but beta2-AR agonist was more potent. Activation of beta-AR receptor transactivates epidermal growth factor receptor (EGFR) and then elicits Akt and ERK1/2 in a PKA-dependent manner, which together up-regulate levels of HIF-1alpha and downstream target genes independently of oxygen level. Our data suggest a novel mechanism in pancreatic cancer cells that links beta-AR and HIF-1alpha signaling under normoxic conditions, with implications for the control of glucose transport, angiogenesis and metastasis.

Amelioration of Cardiac Remodeling in Congestive Heart Failure by β-Adrenoceptor Blockade is Associated with Depression in Sympathetic Activity

This study investigated whether improvement in cardiac function and attenuation of cardiac remodeling by some beta-adrenoceptor (beta-AR) antagonists were associated with a depression in sympathetic activity in congestive heart failure (CHF) due to myocardial infarction (MI). Although cardiac dysfunction, hypertrophy and dilatation as well as increased plasma level of catecholamines are known to occur in CHF, the relationship between these parameters is poorly understood. Three weeks after occlusion of the coronary artery, rats were treated daily with 20 and 75 mg/kg of either atenolol or propranolol for 5 weeks. Sham-operated rats served as controls. Both atenolol and propranolol at 20 and 75 mg/kg doses attenuated the MI-induced cardiac hypertrophy, increases in left ventricular (LV) end-diastolic pressure, LV end-systolic volume and LV end-diastolic volume as well as depressions in LV systolic pressure, LV fractional shortening and cardiac output. PR interval was decreased and QT( c ) interval was increased in CHF; these alterations were ameliorated by both atenolol and propranolol. The increased level of plasma epinephrine in CHF was also depressed by both low and high doses of atenolol and propranolol whereas the increased level of plasma norepinephrine was reduced by high but not low doses of these drugs. The results indicate that the beneficial effects of beta-AR antagonists on cardiac remodeling and heart dysfunction in CHF may be due to the blockade of beta-ARs in the myocardium and a depression in the sympathetic activity.

The β3-Adrenoceptor Agonist GW427353 (Solabegron) Decreases Excitability of Human Enteric Neurons via Release of Somatostatin

beta3 Adrenoceptor (beta3-AR) is expressed on adipocytes and enteric neurons. GW427353 is a human selective beta3-AR agonist with visceral analgesic effects. Some of its effects may involve release of somatostatin (SST) and actions on enteric neurons. The aim of this study was to investigate the mode of action of GW427353 in human submucous neurons. Voltage sensitive dye imaging was used to record from human submucous neurons. SST release from human primary adipocytes was measured with enzyme-linked immunoabsorbent assay. Immunohistochemistry was used to detect adiponectin, beta3-AR, SST, SST2 receptors, tyrosine hydroxylase (TH), and protein gene product 9.5. Confocal imaging showed cytoplasmic beta3-AR labeling in somata of submucous neurons and nerve varicosities. GW427353 had no direct postsynaptic actions but decreased fast synaptic input to submucous neurons. Tissue perfusion with GW427353 reduced nicotine-evoked neuronal spike frequency, an effect prevented by the beta3-AR antagonist SR-59230 and the SST2-receptor antagonist CYN154806 and mimicked by the SST2 receptor agonist octreotide. Adipocytes expressed adiponectin, beta3-AR, and SST. TH-positive fibers were in close proximity to adipocytes. Submucous neurons expressed SST2 receptors. Human primary adipocytes released SST in response to GW427353 in a concentration-dependent manner, an effect abolished by SR-59230. Inhibitory action of GW427353 involves release of SST which stimulates inhibitory SST2 receptors on human submucous neurons. Adipocytes are a potential source for SST. beta3-AR activation may be a promising approach to reduce enteric neuron hyperexcitability. The action of GW427353 may be the neurophysiologic correlate of its beneficial effect in patients with irritable bowel syndrome.

I037 Impaired beta-adrenoceptor-mediated-vasorelaxation to celiprolol is improved by candesartan cilexetil treatment in spontaneously hypertensive rats

Celiprolol, a beta-blocker of third generation, is known to induce beta2-adrenoceptors (-AR) -mediated vasodilation, but the role of other beta-AR subtypes has not been investigated. Thus, the first purpose of this study was to evaluate the participation of beta3-AR in the celiprolol-induced relaxation in Wistar Kyoto rat (WKY) thoracic aorta. Furthermore, the second objective was designed to determine whether the celiprolol-induced relaxation was altered in spontaneously hypertensive rat (SHR) aorta, and to evaluate the effect of systemic or local angiotensin II (AngII) AT1 receptor (AT1R) antagonism on the relaxant effect of celiprolol. Aortic rings were mounted in organ baths and preconstricted with phenylephrine. Cumulative concentration response curves to celiprolol (0.1 to 300 μm) were established in different experimental conditions. Celiprolol produced a significant concentration dependent relaxation in WKY aorta (Emax=66.85±5.4 %, n=10) which was decreased in the presence of nadolol (beta1/beta2-AR antagonist, 10 μm) or SR59230A (beta3-AR antagonist, 1 μm). Relaxation to celiprolol was greatly decreased in denuded aortic rings, and abolished in intact rings pre-treated with L-Name (NOsynthases inhibitor, 100 μm). In SHR aorta, celiprolol-induced relaxation was significantly reduced (Emax=39.4±4.1 %, n=8) compared to WKY, whereas endothelium removal did not alter celiprolol-induced vasorelaxation. A 12 days treatment with candesartan cilexetil (AngII AT1R antagonist, 0.37 or 1 mg/kg/day) reduced systolic blood pressure (SBP) in both WKY and SHR, and significantly improved aorta relaxation to celiprolol more in SHR than in WKY (WKY : Emax=71.7±5,1 %, n=12 ; SHR : Emax=64.2±3.9 %, n=14). Furthermore, local aortic AT1R antagonism with candesartan CV11974 (100 mM) improved celiprolol-induced relaxation, but only in WKY aorta (Emax=78±5.6 %, n=7). Celiprolol induced an endothelial beta1-, beta2-and beta3-AR relaxation in WKY aorta through NO pathway. In SHR aorta, we showed, for the first time, that the celiprolol-induced vasorelaxation was impaired and that only the systemic AT1R antagonism with candesartan cilexetil improved the celiprolol-induced relaxation. This indicated that the beneficial effect of candesartan cilexetil was likely related to decreased SBP but not to specific mechanisms that may occur in aortic smooth muscle and/or endothelial cells.

β2‐adrenoceptors are critical for antidepressant treatment of neuropathic pain

Tricyclic antidepressants (TCAs) are one of the first-line pharmacological treatments against neuropathic pain. TCAs increase the extracellular concentrations of noradrenaline and serotonin by blocking the reuptake transporters of these amines. However, the precise downstream mechanism leading to the therapeutic action remains identified. In this work, we evaluated the role of adrenergic receptors (ARs) in the action of TCAs. We used pharmacological and genetic approaches in mice to study the role of ARs in the antiallodynic action of the TCA nortriptyline. Peripheral neuropathy was induced by the insertion of a polyethylene cuff around the main branch of the sciatic nerve. The specific role of beta(2)-AR was evaluated by studying beta(2)-AR(-/-) mice. We used von Frey filaments to assess mechanical allodynia. The antiallodynic action of nortriptyline was not affected by cotreatment with the alpha(2)-AR antagonist yohimbine, the beta(1)-AR antagonists atenolol or metoprolol, or the beta(3)-AR antagonist SR 59230A. On the contrary, the beta-AR antagonists propranolol or sotalol, the beta(1)/beta(2)-AR antagonists alprenolol or pindolol, or the specific beta(2)-AR antagonist ICI 118,551 blocked the action of nortriptyline. The effect of nortriptyline was also totally absent in beta(2)-AR-deficient mice. Stimulation of beta(2)-AR is necessary for nortriptyline to exert its antiallodynic action against neuropathic pain. These findings provide new insight into the mechanism by which antidepressants alleviate neuropathic pain. Our results also raise the question of a potential incompatibility between beta-blockers that affect beta(2)-AR and antidepressant drugs in patients treated for neuropathic pain.

Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers

BackgroundStress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.Methods and FindingsMigratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.ConclusionsThis work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.

Postischemic Brain Injury Is Attenuated in Mice Lacking the β2-Adrenergic Receptor

Several beta-adrenergic receptor (betaAR) antagonists have been shown to have neuroprotective effects against cerebral ischemia. However, clenbuterol, a beta(2)AR agonist, was shown to have neuroprotective activity by increasing nerve growth factor expression. We used beta(2)AR knockout mice and a beta(2) selective antagonist to test the effect of loss of beta(2)ARs on outcome from transient focal cerebral ischemia. Ischemia was induced by the intraluminal suture method, for 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Neurological score was determined at 24 h reperfusion and infarct size was determined by cresyl violet or 2,3,5-triphenyltetrazolium chloride staining. beta(2)AR knockout mice and wild-type congenic FVB/N controls were studied, as well as 2 groups of wild type mice given either ICI 118,551 (0.2 mg/kg) or 0.9% saline intraperitoneally 30 min before MCAO (n = 10 per group). Changes in expression of heat shock protein (Hsp)72 after ischemia were examined by immunohistochemistry and western blots. Compared with wild type littermates, infarct volume was decreased by 22.3% in beta(2)AR knockout mice (39.7 +/- 10.7 mm(3) vs 51.0 +/- 11.4 mm(3), n = 10/group, P = 0.034) after 60 min of MCAO followed by 24 h reperfusion. Pretreatment with a beta(2)AR selective antagonist, ICI 118,551, also decreased infarct size significantly, by 25.1%, compared with the saline control (32.8 +/- 11.9 mm(3) vs 43.8 +/- 10.3 mm(3), n = 10/group, P = 0.041). Neurological scores were also significantly improved in mice lacking the beta(2)AR or pretreated with ICI 118,551. After cerebral ischemia, total levels of Hsp72 and the number of Hsp72 immunopositive cells were greater in mice lacking beta(2) AR. Brain injury is reduced and neurological outcome improved after MCAO in mice lacking the beta(2)AR, or in wild type mice pretreated with a selective beta(2)AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of beta(2)AR activation in cerebral ischemia. Protection is associated with higher levels of Hsp72, a known antideath protein. The effect of beta(2)AR signaling in the setting of cerebral ischemia is complex and warrants further study.

Prevention of pancreatic cancer by the beta-blocker propranolol

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer deaths and is unresponsive to existing therapy. Smoking and alcohol-induced pancreatitis are among the risk factors for PDAC. We have previously reported that beta-adrenergic receptors (beta-ARs) stimulate the proliferation and migration of human PDAC cells in vitro by cAMP-dependent signaling and that the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates this pathway directly in vitro while additionally stimulating the release of noradrenaline/adrenaline by binding to alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) in hamsters. In this study, we have tested the hypothesis that the beta-AR antagonist propranolol prevents the development of PDAC induced in hamsters with ethanol-induced pancreatitis by NNK. We found that propranolol had strong cancer preventive effects in this animal model. Western blots of pancreatic duct cells and PDAC cells harvested by laser capture microscopy showed significant upregulation of the alpha7 nAChR associated with significant inductions of p-CREB, p-ERK1/2, and increases in epidermal growth factor and vascular endothelial growth factor in PDAC cells of hamsters not treated with propranolol. These effects were reversed by treatment with propranolol. Our data suggest that propranolol may prevent the development of PDAC by blocking cAMP-dependent intracellular signaling, cAMP-dependent release of epidermal growth factor, and PKA-dependent release of vascular endothelial growth factor while additionally downregulating the alpha7 nAChR by inhibiting cAMP-mediated subunit assembly. We conclude that increased cAMP signaling is an important factor that drives the development and progression of PDAC and that the inhibition of cAMP formation is a promising new target for the prevention and adjuvant therapy of PDAC.

Oestrogen changed cardiomyocyte contraction and β‐adrenoceptor expression in rat hearts subjected to ischaemia–reperfusion

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, oestrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and oestrogen replacement on ventricular myocyte contractile function and expression of beta-adrenoceptors (beta-ARs). Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham operation (Sham). A subgroup of OVX rats received oestrogen (E2) replacement (40 microg kg(-1) day(-1)) for 4 weeks. Cardiomyocyte shortening was evaluated in basal conditions and in the presence of isoprenaline (ISO). The expression of beta-ARs was assessed by Western blotting. The presence of lactate dehydrogenase (LDH) activity in the coronary effluent was determined. Ovariectomy promoted body weight gain associated with reduced serum E2 and uterine weight, all of which were abolished by treatment with E2. Ovariectomy increased the amplitude of both basal and ISO-stimulated contractions, increased LDH release, upregulated beta1-AR expression and downregulated beta2-AR expression, all of which were restored by treatment with E2. A beta1-AR antagonist, CGP20712A, but not a beta2-AR antagonist, ICI118,551, significantly decreased the amplitude of ventricular myocyte shortening. Oestrogen decreased cardiomyocyte contraction and the expression of beta1-AR, and increased expression of beta2-AR, and all these effects were abolished by the E2 receptor antagonist, ICI182,780. These data suggest that oestrogen plays a cardioprotective role in female rat hearts subjected to ischaemia-reperfusion injury, and the effects of oestrogen are associated with decreased cardiomyocyte contraction and expression of beta1-AR, and increased expression of beta2-AR.

β1‐ and β2‐Adrenoceptors in basolateral nucleus of amygdala and their roles in consolidation of fear memory in rats

It is known that beta-adrenoceptor (AR) in the basolateral nucleus of amygdala (BLA) plays an essential role in fear memory formation. However, the cellular and subcellular distributions of beta1- and beta2-ARs in the BLA and their roles in fear memory formation are poorly understood. Here, we report that both beta1- and beta2-ARs are predominantly expressed in BLA neurons but not in astrocytes. beta1-AR is distributed in the cell membrane and cytoplasm of neurons, whereas beta2-AR is localized not only in the cell membrane and cytoplasm but also in the nucleus. Intra-BLA infusion of the beta1-AR antagonist metoprolol and atenolol or the beta2-AR antagonist ICI118551 and butoxamine produces a severe deficit in 24-h auditory fear memory, leaving 1-h memory intact. Western-blot analysis reveals that the protein level of cytoplasmic beta1-AR significantly increases 2- and 4-h postconditioning, whereas that of cytoplasmic or nuclear beta2-AR is unchanged. The present results indicate that beta1- and beta2-ARs in the BLA have differential subcellular localizations and both are required for the consolidation of auditory fear memory.

β3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

The rush to adrenaline: drugs in sport acting on the β‐adrenergic system

Athletes attempt to improve performance with drugs that act on the beta-adrenergic system directly or indirectly. Of three beta-adrenoceptor (AR) subtypes, the beta(2)-AR is the main target in sport; they have bronchodilator and anabolic actions and enhance anti-inflammatory actions of corticosteroids. Although demonstrable in animal experiments and humans, there is little evidence that these properties can significantly improve performance in trained athletes. Their actions may also be compromised by receptor desensitization and by common, naturally occurring receptor mutations (polymorphisms) that can influence receptor signalling and desensitization properties in individuals. Indirectly acting agents affect release and reuptake of noradrenaline and adrenaline, thereby influencing all AR subtypes including the three beta-ARs. These agents can have potent psychostimulant effects that provide an illusion of better performance that does not usually translate into improvement in practice. Amphetamines and cocaine also have considerable potential for cardiac damage. beta-AR antagonists (beta-blockers) are used in sports that require steadiness and accuracy, such as archery and shooting, where their ability to reduce heart rate and muscle tremor may improve performance. They have a deleterious effect in endurance sports because they reduce physical performance and maximum exercise load. Recent studies have identified that many beta-AR antagonists not only block the actions of agonists but also activate other (mitogen-activated PK) signalling pathways influencing cell growth and fate. The concept that many compounds previously regarded as 'blockers' may express their own spectrum of pharmacological properties has potentially far-reaching consequences for the use of drugs both therapeutically and illicitly.