Physiological Temperature Changes Fine-Tune ß 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Gal-3 (Galectin-3) and KCa3.1 Mediate Heterogeneous Cell Coupling and Myocardial Fibrogenesis Driven by ßAR (ß-Adrenoceptor) Activation.

Heart failure is associated with sympatho-ßAR (ß-adrenoceptor) activation and cardiac fibrosis. Gal-3 (galectin-3) and KCa3.1 channels that are upregulated in diverse cells of diseased heart are implicated in mediating myocardial inflammation and fibrosis. It remains unclear whether Gal-3 interacts with KCa3.1 leading to cardiac fibrosis in the setting of ßAR activation. We tested the effect of KCa3.1 blocker TRAM-34 on cardiac fibrosis and inflammation in cardiac-restricted ß2-TG (ß2AR overexpressed transgenic) mice and determined KCa3.1 expression in ß2-TG×Gal-3-/- mouse hearts. Mechanisms of KCa3.1 in mediating Gal-3 induced fibroblast activation were studied ex vivo. Expression of Gal-3 and KCa3.1 was elevated in ß2-TG hearts. Gal-3 gene deletion in ß2-TG mice decreased KCa3.1 expression in inflammatory cells but not in fibroblasts. Treatment of ß2-TG mice with TRAM-34 for 1 or 2 months significantly ameliorated cardiac inflammation and fibrosis and reduced Gal-3 level. In cultured fibroblasts, Gal-3 upregulated KCa3.1 expression and channel currents with enhanced membrane potential and Ca2+ entry through TRPV4 (transient receptor potential V4) and TRPC6 (transient receptor potential C6) channels leading to fibroblast activation. In conclusion, ßAR stimulation promotes Gal-3 production that upregulates KCa3.1 channels in noncardiomyocyte cells and activates KCa3.1 channels in fibroblasts leading to hyperpolarization of membrane potential and Ca2+ entry via TRP channels. Gal-3-KCa3.1 signaling mobilizes diverse cells facilitating regional inflammation and fibroblast activation and hence myocardial fibrosis.

Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.

Assessment of Beta-AR protein expression on tumour tissues might be a plausible strategy to select cancer patients who can benefit from Beta-blockers therapy. The aim of this study is to evaluate the differences between resected tissue specimens from primary lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SCC)) in terms of expression pattern of Beta1- and Beta2-AR in both tumour and adjacent surrounding non-tumour tissue. This retrospective study was based on the analysis of 80 patients with histologically confirmed diagnosis of primary Non-Small Cell Lung Cancer (NSCLC) who received surgical treatment. The cases were carefully selected in order to obtain the most homogeneous sample in terms of histologic subtype (40 ADCs and 40 SCCs) and clinical stage (10 each). Beta1- and Beta2-AR expression was determined by immunohistochemistry and the staining evaluated by semi-quantitative scoring using the H-score method. In our NSCLC series, Beta1- and Beta2-AR are differentially expressed. Beta1-AR expression is present at low levels in both SCC and ADC. Likewise, when compared with the matched surrounding non-tumour tissues, Beta1-AR expression level was significantly lower in both histologic subtypes. Conversely, Beta2-AR is highly expressed in both histologic subtypes, but clearly highly expressed in ADC when compared with SCC and with their matched surrounding non-tumour tissue. Overall, this clinicopathological study highlights the differential expression of Beta1- and Beta2-AR in ADC and SCC. Repurposing non-selective Beta-blockers in oncologic setting might be a suitable therapeutic strategy for lung ADC. Graphical abstract.

Chronic stress promotes gastric cancer progression and metastasis: an essential role for ADRB2.

An increasing number of studies indicate that adrenergic signalling plays a fundamental role in chronic stress-induced tumour progression and metastasis. However, its function in gastric cancer (GC) and its potential mechanisms remain unknown. The expression levels of ß-adrenergic receptor (ADRB) in GC cell lines were examined by using real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of ß2 adrenergic receptor (ADRB2) activation and blockade were investigated in vitro in GC cells by using proliferation, migration, invasion, cell cycle and apoptosis assays. Chronic restraint stress (CRS) increased the plasma levels of catecholamines and cortisol and also induced progression and metastasis of GC in vivo. Furthermore, immunohistochemical staining and a TUNEL assay were employed to observe the regulation of cell viability in vivo. The expression levels of ADRB2 in 100 human GC samples were measured by RT-PCR and immunohistochemistry. The stress hormones epinephrine and norepinephrine significantly accelerated GC cell proliferation, invasion and viability in culture, as well as tumour growth in vivo. These effects were reversed by the ADRB antagonists propranolol and ICI118,551 (an ADRB2-specific antagonist). Moreover, the selective ADRB1 antagonist atenolol had almost no effect on tumour cell proliferation and invasion in vitro and in vivo. ADRB2 antagonists suppressed proliferation, invasion and metastasis by inhibiting the ERK1/2-JNK-MAPK pathway and transcription factors, such as NF-κB, AP-1, CREB and STAT3. Analysis of xenograft models using GC cells revealed that ADRB2 antagonists significantly inhibited tumour growth and metastasis, and chronic stress antagonized these inhibitory effects. In addition, chronic stress increased the expression of VEGF, MMP-2, MMP-7 and MMP-9 in transplanted tumour tissue, and catecholamine hormones enhanced the expression of metastasis-related proteins. The expression of ADRB2 was upregulated in tumour tissues and positively correlated with tumour size, histological grade, lymph node metastasis and clinical stage in human GC samples. Stress hormone-induced activation of the ADRB2 signalling pathway plays a crucial role in GC progression and metastasis. These findings indicate that ADRB2 signalling regulates GC progression and suggest ß2 blockade as a novel strategy to complement existing therapies for GC.

CCL2 Induces the Production of ß2 Adrenergic Receptors and Modifies Astrocytic Responses to Noradrenaline.

The decline in brain noradrenaline levels is associated with the progression of certain neurodegenerative diseases. This seems to be due, at least in part, to the ability of noradrenaline to limit glial activation and to reduce the damage associated with it. Our previous studies of the mechanisms involved in this process indicate that noradrenaline induces the production of the chemokine CCL2 in astrocytes. While CCL2 can protect neurons against certain injuries, its overproduction has also proven to be harmful and to prevent noradrenaline neuroprotective effects. Therefore, in this study, we analyze if the modifications caused to astrocytes by an excessive production of CCL2 may alter their response to noradrenaline. Using primary cultures of rat cortical astrocytes, we observed that CCL2 enhances the production of beta 2 adrenergic receptors in these cells. While this potentiates noradrenaline signaling through cAMP, the activation of the transcription factor CREB is inhibited by CCL2. Furthermore, although CCL2 potentiates noradrenaline induction of glycogenolysis, this does not translate into an augmented release of lactate, one of the processes through which astrocytes help support neurons. Additionally, other neuroprotective actions of noradrenaline, such as the production of brain derived neurotrophic factor and the inhibition of the inducible nitric oxide synthase in astrocytes were modified by CCL2. These data suggest that some of the central nervous system alterations related to CCL2 could be due to its effects on adrenergic receptors and its interference with noradrenaline signaling.

Cell-Free Expression, Purification, and Characterization of the Functional ß2-Adrenergic Receptor for Multianalyte Detection of ß-Agonists.

Large-scale expression of ß2-adrenergic receptor (ß2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused ß-adrenergic agonists (ß-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional ß2-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine ß2-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified ß2-AR for ß-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of ß2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting ß-agonist residues.

VCP/p97 regulates ß2AR quality control during receptor biosynthesis.

GPCRs form signalling complexes with other receptors as part of dimers, G proteins and effector partners. A proteomic screen to identify proteins that associate with the ß2-adrenergic receptor (ß2AR) identified many of components of the Endoplasmic-Reticulum-Associated Degradation (ERAD) quality control system [1], including the valosin-containing protein (VCP/p97). Here, we validated the interaction of VCP with co-expressed FLAG-ß2AR, demonstrating, using an inducible expression system, that the interaction of FLAG-ß2AR and VCP is not an artifact of overexpression of the ß2AR per se. We knocked down VCP and noted that levels of FLAG-ß2AR were increased in cells with lower VCP levels. This increase in the level of FLAG-ß2AR did not lead to an increase in the level of functional receptor observed at the cell surface. Similarly, inhibition of the proteasome lead to a dramatic increase in the abundance of TAP-ß2AR, while cellular responses again remained unchanged. Taken together, our data suggests that a substantial proportion of the ß2AR produced is non-functional and VCP plays a key role in the maturation and trafficking of the ß2AR as part of the ERAD quality control process.

Tracking GPCR biosynthesis and degradation using a nonradioactive pulse chase methodology.

The ß2-adrenergic receptor (ß2AR) is a prototypical member of the G protein-coupled receptor (GPCR) superfamily of proteins and is one of the best-characterized GPCRs due to its role in several important physiological systems. Because of limited availability of high quality antibodies against GPCRs, much of the work done on ß2AR took advantage of heterologous expression systems. Overexpressed proteins may overwhelm the cellular regulatory machinery leading potentially to responses distinct from the native protein. To address this issue we generated a stable cell line with a tetracycline-inducible ß2AR tagged with a FLAG epitope, such that we are able to control the quantity of receptor produced. This allows us to induce a discrete pulse of FLAG-ß2AR transcription and translation allowing us to follow the complete life cycle of the protein from synthesis as an immature protein to degradation. We show that such limited pulses of receptor expression lead to signaling phenotypes that more closely reflect endogenous signaling events.

ß2-adrenogenic signaling regulates NNK-induced pancreatic cancer progression via upregulation of HIF-1α.

Cigarette smoking is a risk factor for pancreatic cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, we show that smoking, HIF-1α expression and ß2-adrenogenic receptor (ß2-AR) expression are negatively correlated with the overall survival of pancreatic cancer patients. Moreover, HIF-1α expression and ß2-AR expression are positively correlated with smoking status, different histological differentiation and among the tumor node metastasis (TNM) stages in pancreatic cancer patients. NNK increases HIF-1α expression in pancreatic cancer in vitro and in vivo. Furthermore, knockdown of HIF-1α and ICI118, 551 (a ß2-AR selective antagonist) abrogates NNK-induced pancreatic cancer proliferation and invasion in vitro and inhibits NNK-induced pancreatic cancer growth in vivo. However, using CoCl2 (a HIF-1α stabilizing agent which decreases HIF-1α degradation under normoxia conditions) reverses ICI118, 551 induced effects under NNK exposure. Thus, our data indicate that ß2-AR signaling regulates NNK-induced pancreatic cancer progression via upregulation of HIF-1α. Taken together, ß2-AR signaling and HIF-1α may represent promising therapeutic targets for preventing smoking induced pancreatic cancer progression.

A Her2-let-7-ß2-AR circuit affects prognosis in patients with Her2-positive breast cancer.

BACKGROUND: Our previous studies show that ß2-adrenergic receptor (ß2-AR) is highly expressed in most Her2-overexpressing breast cancers. However, the mechanisms underlying upregulation of the ß2-AR expression in Her2-overexpressing breast cancer cells are not fully understood. The clinical significance of the ß2-AR overexpression in breast cancer is unclear. METHODS: Human breast cancer cells MCF-7 and MCF-7/Her2 were transfected with the let-7 mimics or inhibitors. The expression of ß2-AR was analyzed by Western blot. The ß2-AR status in primary and metastatic sites of breast cancer and the human breast cancer tissue microarrays containing 49 primary tumors and 50 metastatic lymph node tissues was analyzed by immunohistochemistry. The correlation of lymph node metastasis with the ß2-AR level was determined in 59 primary tumor tissues from the patients with Her2-positive breast cancer. The clinical prognostic significance of the ß2-AR overexpression in the patients with Her2-positive breast cancers was evaluated by a retrospective study. RESULTS: The let-7f level in Her2-overexpressing breast cancer cells SKBR3 and BT474 was significantly lower than that in MCF-7 cells, which express low level of Her2. Ectopic expression of Her2 in MCF-7 cells (MCF-7/Her2) represses the expression of microRNA let-7f, which is previously identified to regulate baseline ß2-AR expression. The treatment with MEK1/2 inhibitors PD98059 or PD184352 effectively restored the let-7f level, suggesting that Her2-overexpression-mediated ERK constitutive activation inhibited let-7f, leading to the upregulation of the ß2-AR expression. The transfection with the let-7f mimics markedly downregulated the ß2-AR level, whereas the let-7 inhibitor significantly upregulated the ß2-AR expression in both parental MCF-7 and MCF-7/Her2 cells. In addition, treatment of MCF-7/Her2 cells with isoproterenol resulted in a concentration-dependent reduction of the let-7f expression, demonstrating that the inhibitory effect of Her2 overexpression on let-7f can be reinforced by agonist-triggered ß2-AR activation. We further demonstrate that high level of ß2-AR associates with lymph node metastasis and poor outcome in the patients with Her2-positive breast cancer. CONCLUSIONS: The mutual and reciprocal interaction between Her2, ß2-AR, and let-7f may maintain a high level of ß2-AR in breast cancer cells. Our data suggest that ß2-AR may be a new useful biomarker for predicting prognosis in Her2-positive breast cancer and may also be a promising selective therapeutic target for the aggressive subtype of breast cancer.

Expression of the beta-2 adrenergic receptor (ADRB-2) in human and monkey ovarian follicles: a marker of growing follicles?

BACKGROUND: ADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary. METHODS: We studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined. RESULTS: Immunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 µm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles. CONCLUSIONS: The results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary.

Differential ß2-adrenergic receptor expression defines the phenotype of non-tumorigenic and malignant human breast cell lines.

Breast cancer is the most frequent malignancy in women. Several reports demonstrated that adrenergic receptors (ARs) are involved in breast cancer. Here we observed that epinephrine (Epi), an endogenous AR agonist, caused opposite effects in non-tumorigenic (MCF-10A and HBL-100) and tumor cells (MCF-7 and MDA-MB-231). Thus, Epi, in non-tumor breast cells, as well as isoproterenol (ß-agonist), in all cell lines, maintained a benign phenotype, decreasing cell proliferation and migration, and stimulating cell adhesion. ß-AR expression and cAMP levels were higher in MCF-10A than in MCF-7 cells. ß2-AR knock-down caused a significant increase of cell proliferation and migration, and a decrease of cell adhesion both in basal and in Iso-stimulated conditions. Coincidently, ß2-AR over-expression induced a significant decrease of cell proliferation and migration, and an increase of cell adhesion. Therefore, ß2-AR is implied in cell phenotype and its agonists or antagonists could eventually complement cancer therapy.

Caveolin contributes to the modulation of basal and ß-adrenoceptor stimulated function of the adult rat ventricular myocyte by simvastatin: a novel pleiotropic effect.

The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system ('pleiotropic effects'). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2+]i) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to ß2-, but not ß1-, adrenoceptor stimulation. Under conditions of ß2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered ß-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.

Differential effects of sympathetic nervous system and hypothalamic-pituitary-adrenal axis on systemic immune cells after severe experimental stroke.

Infectious complications are the leading cause of death in the post-acute phase of stroke. Post-stroke immunodeficiency is believed to result from neurohormonal dysregulation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis. However, the differential effects of these neuroendocrine systems on the peripheral immune cells are only partially understood. Here, we determined the impact of the hormones of the SNS and HPA on distinct immune cell populations and characterized their interactions after stroke. At various time points after cortical or extensive hemispheric cerebral ischemia, plasma cortisone, corticosterone, metanephrine and adrenocorticotropic hormone (ACTH) levels were measured in mice. Leukocyte subpopulations were flow cytometrically analyzed in spleen and blood. To investigate their differential sensitivity to stress hormones, splenocytes were incubated in vitro with prednisolone, epinephrine and their respective receptor blockers. Glucocorticoid receptor (GCR) and beta2-adrenergic receptor (ß2-AR) on leukocyte subpopulations were quantified by flow cytometry. In vivo effects of GCR and selective ß2-AR blockade, respectively, were defined on serum hormone concentrations, lymphopenia and interferon-γ production after severe ischemia. We found elevated cortisone, corticosterone and metanephrine levels and associated lymphocytopenia only after extensive brain infarction. Prednisolone resulted in a 5 times higher cell death rate of splenocytes than epinephrine in vitro. Prednisolone and epinephrine-induced leukocyte cell death was prevented by GCR and ß2-AR blockade, respectively. In vivo, only GCR blockade prevented post ischemic lymphopenia whereas ß2-AR preserved interferon-γ secretion by lymphocytes. GCR blockade increased metanephrine levels in vivo and prednisolone, in turn, decreased ß2-AR expression on lymphocytes. In conclusion, mediators of the SNS and the HPA axis differentially affect the systemic immune system after stroke. Moreover, our findings suggest a negative-feedback of corticosteroids on the sympathetic axis which may control the post-stroke stress-reaction. This complex interplay between the HPA and the SNS after stroke has to be considered when targeting the neurohormonal systems in the post acute phase of severe stroke.

Expression and functions of ß1- and ß2-adrenergic receptors on the bulbospinal neurons in the rostral ventrolateral medulla.

The expression and effects of ß-adrenergic receptors (ß-ARs) on the neurons of the bulbospinal rostral ventrolateral medulla (RVLM) have been limitedly examined to date. The objective of this study was to examine the expression of ß1- and ß2-ARs on the bulbospinal RVLM neurons electrophysiologically and histologically. To directly investigate whether RVLM neurons display sensitivity to metoprolol (a ß1-AR antagonist), dobutamine (a ß1-AR agonist), butoxamine (a ß2-AR antagonist), and salbutamol (a ß2-AR agonist), we examined changes in the membrane potentials of the bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion of these drugs. During metoprolol superfusion, 16 of the 20 RVLM neurons were hyperpolarized, and 5 of the 6 RVLM neurons were depolarized during dobutamine superfusion. During butoxamine superfusion, 11 of the 16 RVLM neurons were depolarized, and all of the 8 RVLM neurons were hyperpolarized during salbutamol superfusion. These results suggest the expression of ß1- and ß2-ARs on the RVLM neurons. To determine the presence of ß1- and ß2-ARs histologically, immunofluorescence examination was performed. Five metoprolol-hyperpolarized neurons were examined for ß1-AR and tyrosine hydroxylase (TH) immunoreactivity. All of the neurons displayed ß1-AR immunoreactivity, whereas three of the neurons displayed TH immunoreactivity. All of the five RVLM neurons that became depolarized during metoprolol superfusion and hyperpolarized during butoxamine superfusion displayed ß1- and ß2-AR immunoreactivity. Our findings suggest that ß1-AR antagonists or ß2-AR agonists may decrease blood pressure through decreasing the activity of the bulbospinal RVLM neurons.

A modified approach to induce predictable congestive heart failure by volume overload in rats.

The model of infrarenal aortocaval fistula (ACF) has recently gained new interest in its use to investigate cardiac pathophysiology. Since in previous investigations the development of congestive heart failure (CHF) was inconsistent and started to develop earliest 8-10 weeks after fistula induction using a 18G needle, this project aimed to induce a predictable degree of CHF within a definite time period using a modified approach. An aortocaval fistula was induced in male Wistar rats using a 16G needle as a modification of the former 18G needle-technique described by Garcia and Diebold. Results revealed within 28 ± 2 days of ACF significantly increased heart and lung weight indices in the ACF group accompanied by elevated filling pressure. All hemodynamic parameters derived from a pressure-volume conductance-catheter in vivo were significantly altered in the ACF consistent with severe systolic and diastolic left ventricular dysfunction. This was accompanied by systemic neurohumoral activation as demonstrated by elevated rBNP-45 plasma concentrations in every rat of the ACF group. Furthermore, the restriction in overall cardiac function was associated with a ß1- and ß2-adrenoreceptor mRNA downregulation in the left ventricle. In contrast, ß3-adrenoreceptor mRNA was upregulated. Finally, electron microscopy of the left ventricle of rats in the ACF group showed signs of progressive subcellular myocardial fragmentation. In conclusion, the morphometric, hemodynamic and neurohumoral characterization of the modified approach revealed predictable and consistent signs of congestive heart failure within 28 ± 2 days. Therefore, this modified approach might facilitate the examination of various questions specific to CHF and allow for pharmacological interventions to determine pathophysiological pathways.

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors.

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.

Expression of ß2 adrenoceptors within enteric neurons of the horse ileum.

The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of ß2-AR in the horse ileum using double-immunofluorescence. The ß2-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing ß2-AR-IR. Since ß2-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near ß2-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system.

The Arf GAP AGAP2 interacts with ß-arrestin2 and regulates ß2-adrenergic receptor recycling and ERK activation.

AGAP2 [Arf (ADP-ribosylation factor) GAP (GTPase-activating protein) with GTP-binding-protein-like, ankyrin repeat and PH (pleckstrin homology) domains] is a multidomain Arf GAP that was shown to promote the fast recycling of transferrin receptors. In the present study we tested the hypothesis that AGAP2 regulates the trafficking of ß2-adrenergic receptors. We found that AGAP2 formed a complex with ß-arrestin1 and ß-arrestin2, proteins that are known to regulate ß2-adrenergic receptor signalling and trafficking. AGAP2 co-localized with ß-arrestin2 on the plasma membrane, and knockdown of AGAP2 expression reduced plasma membrane association of ß-arrestin2 upon ß2-adrenergic receptor activation. AGAP2 also co-localized with internalized ß2-adrenergic receptors on endosomes, and overexpression of AGAP2 slowed accumulation of ß2-adrenergic receptor in the perinuclear recycling endosomes. In contrast, knockdown of AGAP2 expression prevented the recycling of the ß2-adrenergic receptor back to the plasma membrane. In addition, AGAP2 formed a complex with endogenous ERK (extracellular-signal-regulated kinase) and overexpression of AGAP2 potentiated ERK phosphorylation induced by ß2-adrenergic receptors. Taken together, these results support the hypothesis that AGAP2 plays a role in the signalling and recycling of ß2-adrenergic receptors.