Validation of Fenoterol to Study β2-Adrenoceptor Function in the Rat Urinary Bladder

Fenoterol is a β₂-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by β₂-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via β₃-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study β₂-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC₅₀ 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the β₂-AR antagonist ICI 118,551 and the β₃-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC₅₀ 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC₅₀ 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess β₂-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the β-AR.

The role of the autonomic nervous system in stress cardiomyopathy

Aim. To identify the role of the autonomic nervous system in stress cardiomyopathy in an experimental model of Takotsubo syndrome.Materials and methods. The study was carried out on 120 female Wistar rats. Stress modeling was performed by immobilizing animals on the back for 24 hours. Intact rats were used as controls. The rats were decapitated after termination of immobilization under general anesthesia with ether. Stress cardiomyopathy (SCM) was quantified by accumulation of 99mTc pyrophosphate radiopharmaceutical (99mTc PP) in the myocardium. The pharmacological agents used included the ganglionic blocker hexamethonium, administered five times at a dose of 20 mg / kg; guanethidine (50 mg / kg) administered subcutaneously once a day for three days, the last injection was performed 24 hours before immobilization; the muscarinic receptor antagonist atropine methyl nitrate (1 mg / kg); the α1-AR (adrenergic receptor) antagonist prazosin (2 mg / kg); the α2-AR antagonist yohimbine, administered at a dose of 2 mg / kg; the β1-AR antagonist nebivolol (1.2 mg / kg); the β2-AR antagonist ICI 118,551 (0.3 mg / kg); and the β3-AR antagonist L-748337 (0.1 mg / kg).Results. Three-day administration of guanethidine caused a decrease in the degree of 99mTc-PP accumulation in the heart by 35.9%. Hexamethonium did not affect the degree of SCM. The blockade of the muscarinic receptor caused an increase in accumulation of 99mTc-PP by 26.5%. Inhibition of α1-AR did not affect SCM. The blockade of α2-AR caused a 2.2-fold increase in the accumulation compared with stress control. The blockade of β1-AR reduced 99mTc-PP accumulation by 2.5 times. The blockade of β2-AR by ICI 118,551 increased the degree of 99mTcPP accumulation by 34.6%. Inhibition of β3-AR had no effect on SCM.Conclusion. The adrenergic system and β1-adrenergic receptor play an important role in the development of SCM. The parasympathetic nervous system ensures resistance of the heart to stress.

Serotonin after β-Adrenoreceptors’ Exposition: New Approaches for Personalized Data in Breast Cancer Cells

Serotonin is an important monoamine in the human body, playing crucial roles, such as a neurotransmitter in the central nervous system. Previously, our group reported that β-adrenergic drugs (ICI 118,551, isoprenaline, and propranolol) influence the proliferation of breast cancer cells (MCF-7 cells) and their inherent production of adrenaline. Thus, we aimed to investigate the production of serotonin in MCF-7 cells, clarifying if there is a relationship between this production and the viability of the cells. To address this question, briefly, we treated the MCF-7 cells with ICI 118,551, isoprenaline, and propranolol, and evaluated cellular viability and serotonin production by using MTT, Sulforhodamine B (SRB) and Neutral Red (NR) assays, and HPLC-ECD analysis, respectively. Our results demonstrate that isoprenaline promotes the most pronounced endogenous synthesis of serotonin, about 3.5-fold greater than control cells. Propranolol treatment also increased the synthesis of serotonin (when compared to control). On the other hand, treatment with the drug ICI 118,551 promoted a lower endogenous synthesis of serotonin, about 1.1-fold less than what was observed in the control. Together, these results reveal that MCF-7 cells can produce serotonin, and the drugs propranolol, isoprenaline and ICI 118,551 influence this endogenous production. For the first time, after modulation of the β-adrenergic system, a pronounced cellular growth can be related to higher consumption of serotonin by the cells, resulting in decreased levels of serotonin in cell media, indicative of the importance of serotonin in the growth of MCF-7 cells.

Abstract MP26: Angiotensin-(1-7) Increases Vascular Beta-2 Adrenergic Receptor Expression In Aging Mice

Aging is associated with increased sympathetic tone, which desensitizes vascular beta-2 adrenergic receptors (B2AR) to impair vasodilation and promote hypertension. We recently demonstrated that chronic treatment with angiotensin (Ang)-(1-7), a protective hormone of the renin-angiotensin system, decreases blood pressure and cardiac sympathetic tone in aging mice. In this study, we hypothesized that sympathoinhibitory effects of Ang-(1-7) would restore vascular B2AR expression to lower blood pressure in aging. To test this, aging (16-month-old) and young (2-month-old) male C57BL/6J mice received Ang-(1-7) [400 ng/kg/min, n=4] or saline (n=4) infusion for 6 weeks via subcutaneous osmotic mini-pump. At end of treatment, alpha- and beta-adrenergic receptor gene expression was measured in mesenteric arteries, thoracic aorta, and cardiac tissue by quantitative real-time PCR and quantified with 2-ΔΔCT methods. In a separate experiment, male C57BL/6J mice received a single subcutaneous injection of Ang-(1-7) (2 mg/mg, n=4), saline (n=3), or the B2AR antagonist ICI 118,551 (1 mg/kg) plus Ang-(1-7) (n=3). Blood pressure was measured via a carotid artery catheter at baseline and post-treatment. We found that aging mice have decreased mesenteric B2AR gene expression, which was restored by Ang-(1-7) (young saline: 1.04±0.35; aged saline: 0.46±0.17; aged Ang-(1-7): 1.04±0.31 A.U., p=0.026). As a control, Ang-(1-7) did not induce significant changes in B2AR mRNA in thoracic aorta or cardiac tissue, or changes in other adrenergic receptor subtypes (alpha1 or beta1) in any of the tissues studied in aging mice (p>0.05). We further found that depressor effects of acute Ang-(1-7) in mice are attenuated by B2AR blockade (saline: Δ -11±4 mmHg; Ang-(1-7): Δ -26±3 mmHg; ICI 118,551+Ang-(1-7): Δ -4±4 mmHg, p=0.009). Overall, these findings suggest that Ang-(1-7) restores B2AR expression in mesenteric resistance vessels in aging mice, and depressor effects of acute Ang-(1-7) are partially mediated by B2AR. These data support the concept that Ang-(1-7) decreases blood pressure in aging by restoring B2AR-mediated vasodilation. More broadly, Ang-(1-7) may provide a novel treatment target for age-related hypertension and cardiovascular disease.

Olodaterol exerts anti-inflammatory effects on COPD airway epithelial cells

Background Airway inflammation is a key feature of chronic obstructive pulmonary disease (COPD) and inhaled corticosteroids (ICS) remain the main treatment for airway inflammation. Studies have noted the increased efficacy of ICS and long-acting beta 2 agonist (LABA) combination therapy in controlling exacerbations and improving airway inflammation than either monotherapy. Further studies have suggested that LABAs may have inherent anti-inflammatory potential, but this has not been well-studied. Objective We hypothesize that the LABA olodaterol can inhibit airway inflammation resulting from exposure to respiratory syncytial virus (RSV) via its binding receptor, the β2-adrenergic receptor. Methods Human bronchial epithelial brushing from patients with and without COPD were cultured into air–liquid interface (ALI) cultures and treated with or without olodaterol and RSV infection to examine the effect on markers of inflammation including interleukin-8 (IL-8) and mucus secretion. The cell line NCI-H292 was utilized for gene silencing of the β2-adrenergic receptor via siRNA as well as receptor blocking via ICI 118,551 and butaxamine. Results At baseline, COPD-ALIs produced greater amounts of IL-8 than control ALIs. Olodaterol reduced RSV-mediated IL-8 secretion in both COPD and control ALIs and also significantly reduced Muc5AC staining in COPD-ALIs infected with RSV. A non-significant reduction was seen in control ALIs. Gene silencing of the β2-adrenergic receptor in NCI-H292 negated the ability of olodaterol to inhibit IL-8 secretion from both RSV infection and lipopolysaccharide stimulus, as did blocking of the receptor with ICI 118,551 and butaxamine. Conclusions Olodaterol exhibits inherent anti-inflammatory properties on the airway epithelium, in addition to its bronchodilation properties, that is mediated through the β2-adrenergic receptor and independent of ICS usage.

Radioligand binding assay for the simultaneous determination of β1- and β2-adrenergic receptors in human blood cells

Во многих тканях и клетках человека присутствуют как β1-, так и β2-адренорецепторы, информация о содержании и динамике поведения кото- рых часто является клинически значимой. В настоящем исследовании предложена методика раздельного определения обоих типов адреноре- цепторов на основе радиолигандного анализа с использованием 125I-йодоцианопиндолола, включающая проведение трех измерений: 1) без лигандов-конкурентов; 2) в присутствии селективного лиганда ICI 118,551 (0,25 мкМ); 3) в присутствии двух селективных лигандов ICI 118,551 и CGP 20712 (по 0,25 мкМ каждого). Методика протестирована на модельной системе из двух трансгенных линий клеток с экспрессией реком- бинантных β1- и β2-адренорецепторов. При соотношении количества β1-адренорецепторов к β2-адренорецепторам 1:10 погрешность измере- ния составляет около 15%. Анализ 9 клеточных линий, представляющих различные типы клеток крови, показал наличие β2-адренорецепторов в клетках Daudi, Raji, Dami, K-562, HL-60, U-937 и THP-1 и их отсутствие в Т-лимфоцитарных клетках Jurkat и MOLT-4. β1-адренорецепторы достоверно зарегистрированы лишь в клетках THP-1 моноцитарного происхождения. В остальных клетках, за исключением линии Dami, их количество оказалось ниже порога детекции, оцениваемого на уровне 250 молекул на клетку. Измерения, выполненные на мононуклеарных клетках периферической крови здоровых доноров, продемонстрировали присутствие β2-адренорецепторов в диапазоне от 1000 до 2500 моле- кул на клетку, тогда как содержание β1-адренорецепторов во всех случаях находилось на грани или за гранью порога детекции. По-видимому, изучение β1-адренорецепторов в дальнейшем следует проводить на отдельных фракциях клеток крови, в частности на фракции моноцитов β1- and β2-adrenergic receptors are presented in various human tissues and cells, while the information of their content and dynamic behavior is oftenly considered as clinically significant. In this study, a method for the separate determination of both types of adrenoceptors based on radioligand binding analysis using 125I iodocyanopindolol is proposed, comprising three measurements: 1) without competing ligands; 2) in the presence of selective ligand ICI 118,551 (0.25 μM); 3) in the presence of two selective ligands ICI 118,551 and CGP 20712 (0.25 μM each). The technique was tested on a model system of two transgenic cell lines with the expression of recombinant β1- and β2-adrenergic receptors. If the ratio of the number of β1-adrenergic receptors to β2-adrenergic receptors is 1:10, the measurement error is about 15%. Analysis of 9 cell lines representing different types of blood cells showed the presence of β2-adrenergic receptors in Daudi, Raji, Dami, K-562, HL-60, U-937 and THP-1 cells and their absence in Jurkat and MOLT-4 cells. β1-adrenergic receptors are reliably registered only in THP-1 cells of monocytic origin. In the remaining cell lines, with the exception of Dami, the number of β1-adrenergic receptors was found below the detection limit, estimated as 250 molecules per cell. Measurements performed on the peripheral blood mononuclear cells of healthy donors showed the presence of β2-adrenergic receptors in the range from 1000 to 2500 molecules per cell, while the content of β1-adrenergic receptors in all cases appeared to be on the border or beyond the detection limit. Apparently, further study of β1-adrenergic receptors should be performed on the blood cells isolated fractions, on monocytes in particular

Targeting β2-Adrenergic Receptors Shows Therapeutical Benefits in Clear Cell Renal Cell Carcinoma from Von Hippel–Lindau Disease

Von Hippel–Lindau (VHL), is a rare autosomal dominant inherited cancer in which the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HB), CNS-HB, and clear cell renal cell carcinoma (ccRCC). ccRCC ranks third in terms of incidence and first in cause of death. Standard systemic therapies for VHL-ccRCC have shown limited response, with recurrent surgeries being the only effective treatment. Targeting of β2-adrenergic receptor (ADRB) has shown therapeutic antitumor benefits on VHL-retinal HB (clinical trial) and VHL-CNS HB (in vitro). Therefore, the in vitro and in vivo antitumor benefits of propranolol (ADRB-1,2 antagonist) and ICI-118,551 (ADRB-2 antagonist) on VHL−/− ccRCC primary cultures and 786-O tumor cell lines have been addressed. Propranolol and ICI-118,551 activated apoptosis inhibited gene and protein expression of HIF-2α, CAIX, and VEGF, and impaired partially the nuclear internalization of HIF-2α and NFĸB/p65. Moreover, propranolol and ICI-118,551 reduced tumor growth on two in vivo xenografts. Finally, ccRCC patients receiving propranolol as off-label treatment have shown a positive therapeutic response for two years on average. In summary, propranolol and ICI-118,551 have shown antitumor benefits in VHL-derived ccRCC, and since ccRCCs comprise 63% of the total RCCs, targeting ADRB2 becomes a promising drug for VHL and other non-VHL tumors.