β2-AR Protein Research Articles

Impact of Tumoral β2-Adrenergic Receptor Expression on Chemotherapeutic Response and Prognosis in Patients with Advanced Colorectal Cancer.

The β2-adrenergic receptor (β2-AR) is a therapeutic target for circulatory agonists and exhibits oncogenic activity in several cancers. However, its role in advanced colorectal cancer (CRC) treated using chemotherapy remains unclear. We investigated the potential of β2-AR as a novel chemosensitivity marker and therapeutic target in inoperable CRC. β2-AR expression was evaluated immunohistochemically in 80 advanced or recurrent CRC cases for which untreated resected specimens were available before systemic chemotherapy implementation. We assessed the relationship among β2-AR protein expression, clinicopathological factors, therapeutic response, and prognosis. Furthermore, we evaluated the significance of β2-AR as an in vitro and in vivo therapeutic target using CRC cell lines and a CRC xenograft model treated with the β-blocker, propranolol, and other anticancer agents. High tumoral β2-AR expression was associated with shorter progression-free survival and chemotherapeutic resistance in patients treated with oxaliplatin-based regimens and bevacizumab-based regimens. We found no synergistic effect between propranolol and oxaliplatin. However, combined administration of propranolol and bevacizumab induced significant tumor shrinkage in the CRC xenograft model. β2-AR is a possible biomarker for chemosensitivity and prognosis in advanced CRC. Repositioning existing β-blockers could be beneficial for treating CRC resistant to existing treatment regimens.

Role of autonomic nervous system in BDE-209 maternal exposure induced immunotoxicity in female offspring.

Decabrominated diphenyl ether (BDE-209) is a typical persistent organic pollutant that can cross the placental barrier, increasing the exposure risk for offspring. Norepinephrine (NE) from nerve terminals and acetylcholine (Ach) can bind to specific receptors on immune cells, inhibit the immune function of the body then cause immunotoxicity. However, whether maternal exposure to BDE-209 could lead to immunotoxicity in the offspring by acting on the sympathetic and parasympathetic nervous systems remains unclear. In view of this, the pregnancy and lactation rat BDE-209 exposure model was established and the results demonstrated that pregnancy and lactation BDE-209 exposure could induce immunotoxicity to female offspring via affecting immunopathology (hematological and biochemical parameters, organ indices, and spleen histopathological), decreasing humoral immunity (serum hemolysin, immunoglobulins, and cytokine productions), damaging cellular immunity (splenic lymphocytes and spleen cytokine productions), and restraining nonspecific immunity. Moreover, a dramatically significant correlation was observed between spleen nerve indices and immunity indices. Additionally, the mechanism revealed that maternal BDE-209 exposure caused offspring immunotoxicity through (1) activating MHC/PKCθ/NF-κB pathway; (2) promoting sympathetic nervous pathway, by upregulating the expression of β2AR protein, which in turn elevating cAMP, following activate PKA and phosphorylate CREB, ultimately leading to immunotoxicity;(3) activating parasympathetic nerve pathway by reducing the binding with Ach and α7nAchR, upregulating the expression of JAK2 and phosphorylating STAT3, induced immunotoxicity of female offspring.

A cryptic mode of GPCR regulation revealed.

Over three decades of research have provided thorough insights into G protein-coupled receptor (GPCR) regulation. In a recent issue of Molecular Cell, Fonseca et al. identified a previously overlooked desensitization mechanism. Agonist activation of the β2-adrenoceptor (β2AR) causes its S-nitrosylation that is required for the receptor to internalize and desensitize. Eliminating β2AR S-nitrosylation by mutation of C265 augments β2AR protein kinase A signaling, enables β2AR nitric oxide (NO) signaling, renders mice resistant to bronchoconstriction, and protects mice from allergen-induced asthma.

Expression pattern and clinical significance of beta 2-adrenergic receptor in oral squamous cell carcinoma: an emerging prognostic indicator and future therapeutic target.

Beta 2-AdrenergicReceptor (β2-AR) is significantly overexpressed in various types of malignancies, which is associated with the worst prognosis. However, the role of β2-AR in oral cancer is not well identified. The present study aimed at investigating the β2-AR gene expression and its significance in relation withthe clinicopathological features and overall survival of oral squamous cell carcinoma (OSCC) patients. Immunohistochemistry, western blot and quantitative real-time PCR techniques were used to analyze β2-AR protein and mRNA levels in a total of 65 histopathologically confirmed OSCC tissues (case group) and 65 normal tissues (control group) from the oral cavity. Out of the total of 65 OSCC tissues, 41 tissues (63.1%) exhibited high expression for β2-AR protein. Percent positivity and relative density (mean ± SD) of protein were higher in the case group ascompared to the control group (positivity 40.31 ± 3.01 vs. 20.46 ± 1.93, p < 0.001; density 2.77 ± 1.17 vs. 1.28 ± 0.37, p < 0.001). In addition, β2-AR mRNA level was also upregulated in patients comparedtothe controls (2.36 ± 1.30 vs. 1.09 ± 0.42, p < 0.001) and showed a positive correlation with immunostaining of protein in OSCC (r = 0.48, p = 0.011). High β2-AR protein expression was significantly associated with multiple risk habits (p = 0.045), histological differentiation (p = 0.013), clinical TNM stages (p = 0.014), and poor survival (p = 0.006) of patients. In the Cox proportional hazards model, β2-AR was identified as a prognostic biomarker of OSCC (p = 0.047). β2-AR protein level is identified as an independent significant prognostic factor in patients with oral carcinoma.

Restraint stress induces uterine microenvironment disorder in mice during early pregnancy through the β2-AR/cAMP/PKA pathway

During pregnancy, uterus undergoes the environment adaptation as part of a program of development. In the world, one in four people worldwide suffer from mental illness, especially pregnant women. β-Adrenergic receptor (β-AR) is an important regulator that converts environmental stimuli into intracellular signals in mice uterus. CD-1 (ICR) mice undergone restraint stress, which was a case in model to simulate the psychological stress. The plasma and implantation sites in uterus were obtained and examined. PCR analysis demonstrated that β2-AR expression levels in embryo day (E) 3, 5 and 7 were kept at a significantly higher level (p < 0.05) under restraint stress and higher than β1-AR and β3-AR in different gestation ages. The β2-AR protein levels were obviously increased (p < 0.05) due to the markedly elevated norepinephrine (NE) concentration (p < 0.05). In our previous study, restraint stress can induce the apoptosis and inflammation. Also, the matrix metalloprotein-9 (MMP-9) was decreased significantly (p < 0.05) under restraint stress. Meanwhile, Caspase3, p-NF-κB p65 and p-ERK1/2 were obviously increased (p < 0.05) in the work. In vitro studies showed that the p-ERK1/2 and Caspase-3 levels were raised (p < 0.05) after β2-AR was activated. However, they were decreased when PKA was blocked. The protein levels of Caspase-3 were reduced when ERK and NF-κB were blocked (p < 0.05). In conclusion, the β2-AR/cAMP/PKA pathway promoted apoptosis and affected the development of the uterus through the ERK and NF-κB signaling pathway. The findings of this study may provide evidence for female reproduction under psychological stress.

Beta adrenergic receptor activation inhibits oral cancer migration and invasiveness

ObjectiveThe aim of this study was to verify β2-AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of this receptor in migration and invasion of these neoplastic cells. DesignSCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR. Cell migration and invasion were analyzed by wound healing assay and transwell invasion camera system. Different concentrations (0.1, 1 and 10 μM) of norepinephrine were used to stimulate, and 1 μM propranolol was used to block the beta-adrenergic receptors on cancer cells. Differences in median values of SCC-9 and SCC-25 and β2-AR protein expression were analyzed by Friedman test and in case of significant differences; pairwise comparisons were performed using Bonferroni correction. ResultsThe results showed that the β2-AR gene and protein expression were observed in both oral cancer cell lines. The concentration of 10 μM of norepinephrine significantly inhibited (p ≤ 0.05) migration of SCC-9 and SCC-25 cell lines. Furthermore, there was a significant reduction (p ≤ 0.05) in the effect of norepinephrine on cell migration when the β2-AR was inhibited by propranolol. The blockade by propranolol showed a tendency to reverse the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25. ConclusionsThe use of beta-adrenergic receptor agonists could become an adjuvant therapeutic target in the treatment of this malignancy.

Molecular dynamics simulation involved in expounding the activation of adrenoceptors by sympathetic nervous system signaling

G-protein–coupled receptors are integral membrane proteins involved in signal transduction pathways, making them an appealing drug targets for a wide spectrum of diseases. The previous literature reports provide an evidence that catecholamine regulates metastasis by actuating the β2-adrenergic receptor (β-2AR). Molecular dynamics simulations were carried out for 1000 ns to understand the effect of the catecholamine on the human β-2AR. On comparing the apoprotein structure of β-2AR with that of catecholamine interacted β-2AR protein, a large change in structural assembly is observed in the helical regions which confirm the activation of β-2AR protein. The visualization of internal natural pathway of β-2AR protein structure gives us detailed information about deviation in TM helixes. The compactness of protein structure shows β-2AR protein interacting with epinephrine is much stable than β-2AR protein interacting with norepinephrine structure. The Gibbs free energy shows norepinephrine as a partial agonist whereas epinephrine as full agonist for β-2AR protein.

Structural Insights into the Process of GPCR-G Protein Complex Formation

The crystal structure of the β2-adrenergic receptor (β2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (β2AR-Gsempty). Unfortunately, the β2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the β2AR and GDP-bound Gs protein (β2AR-GsGDP) that may represent an intermediate on the way to the formation of β2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the β2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the β2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.

ADRB2 Gene Polymorphisms and Salbutamol Responsiveness in Serbian Children with Asthma.

Inhaled β2 adrenergic receptor (β2-AR) agonists are the mainstay of asthma therapy. The β2-AR protein is encoded by the ADRB2 gene and variants within this gene can have significant consequences for modulating the response to asthma therapy. This cross-sectional study performed at the University Children’s Hospital in Belgrade, included 54 children with asthma. The subjects were genotyped for ADRB2 +46A>G (Arg16Gly, rs1042713) and +79C>G (Gln27Glu, rs 1042714) polymorphisms and the association with asthma severity and response to inhaled salbutamol was examined. In Serbian asthmatic children, allele +46A was detected with a frequency of 41.7% and allele +79G was detected with a frequency of 23.1%. Allele +46G was found to be associated with a better response to inhaled salbutamol (p <0.05) and with mild form of asthma (p <0.05). Polymorphism ADRB2 +46A>G may be a determinant of asthma severity and response to salbutamol in children with asthma. We did not find any association of +79C>G polymorphisms with the asthma severity and bronchodilator response to inhaled salbutamol. The results of this study can be potentially useful for personalization of asthma treatment.

Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mouse spleen.

The cholinergic anti-inflammatory pathway (CAP) is an innate neural reflex where parasympathetic and sympathetic nerves work jointly to control inflammation. Activation of CAP by vagus nerve stimulation (VNS) has paved way for novel therapeutic strategies in treating inflammatory diseases. Recently, we discovered that VNS mediated splenic acetylcholine (ACh) release and subsequent immunosuppression in response to LPS associated inflammation is impaired in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1) expression, a key enzyme responsible for prostaglandin E2 synthesis. Here, we have further investigated the consequences of mPGES-1 deficiency on various molecular/cellular events in the spleen which is critical for the optimal functioning of VNS in endotoxaemic mice. First, VNS induced splenic norepinephrine (NE) release in both mPGES-1 (+/+) and (-/-) mice. Compared to mPGES-1 (+/+), immunomodulatory effects of NE on cytokines were strongly compromised in mPGES-1 (-/-) splenocytes. Interestingly, while LPS increased choline acetyltransferase (ChAT) protein level in mPGES-1 (+/+) splenocytes, it failed to exert similar effects in mPGES-1 (-/-) splenocytes despite unaltered β2 AR protein expression. In addition, nicotine inhibited TNFα release by LPS activated mPGES-1 (+/+) splenocytes in vitro. However, such immunosuppressive effects of nicotine were reversed both in mPGES-1 (-/-) mouse splenocytes and human PBMC treated with mPGES-1 inhibitor. In summary, our data implicate PGE2 as an important mediator of ACh synthesis and noradrenergic/cholinergic molecular events in the spleen that constitute a crucial part of the CAP immune regulation. Our results suggest a possible link between cholinergic and PG system of CAP that may be of clinical significance in VNS treatment.

Effect of electroacpuncture of Neiguan (PC 6) and Tianquan (PC 2) on skin temperature, blood perfusion, and adrenoceptor response in rats with acute myocardial ischemia

ObjectiveTo investigate the effect of electroacupuncture (EA) of acupoints Neiguan (PC 6) and Tianquan (PC 2) on the skin temperature, blood perfusion, and alpha1- and beta2-adrenoreceptor (α1-AR and β2-AR) protein and mRNA level in rats with acute myocardial ischemia. MethodsThirty male adult Wistar rats [(230 ± 20) g] were randomly assigned into five groups (n = 6 each): a control group, sham operation group, model group, model group treated with EA at low frequency (L-EA, 2 Hz, 1 mA) and model group treated with EA at high frequency (H-EA, 100 Hz, 1 mA). The rat model was prepared by ligating the left anterior descending coronary artery. Electroacupuncture was performed at the left Neiguan (PC 6) for 20min daily for 3 d. After the 3rd time of the treatment, measurements of skin blood perfusion and temperature in Neiguan (PC 6) and Tianquan (PC 2) in all groups were made by laser speckle contrast imaging and infrared thermal image instrument. Real-time PCR and ELISA were used to measure mRNA level and protein level of α1-AR and β2-AR in the skin tissues of Neiguan (PC 6) and Tianquan (PC 2), respectively. Serum levels of cTnT and electrocardiogram were used to identify the state of myocardium. ResultsIn the group receiving electroacupunture at Neiguan (PC 6), compared with control group, the skin temperature, blood perfusion, and β2-AR mRNA and protein level of model group significantly decreased (P < 0.05), α1-AR mRNA and protein level of model group significantly increased (P < 0.05); compared with model group, the skin temperature, blood perfusion, and β2-AR mRNA and protein level of L-EA significantly increased (P < 0.05), α1-AR mRNA and protein level of L-EA and H-EA significantly decreased (P < 0.05). The same trend has taken place in the former four groups of the Tianquan (PC 2). ConclusionLow-or high-frequency electroacupuncture can improve the skin temperature and blood perfusion which may be induced by decreasing the level of α1-AR and increasing the level of β2-AR of the Neiguan (PC 6) and Tianquan (PC 2) in the rat with acute myocaidial ischemia.

Trends in Severe Obesity Among 23 Million U.S. Children Aged 2–4 Years Who Enrolled in WIC — United States, 2000–2014

The aim of this study was to verify β2-AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of this receptor in migration and invasion of these neoplastic cells.SCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR. Cell migration and invasion were analyzed by wound healing assay and transwell invasion camera system. Different concentrations (0.1, 1 and 10 μM) of norepinephrine were used to stimulate, and 1 μM propranolol was used to block the beta-adrenergic receptors on cancer cells. Differences in median values of SCC-9 and SCC-25 and β2-AR protein expression were analyzed by Friedman test and in case of significant differences; pairwise comparisons were performed using Bonferroni correction.The results showed that the β2-AR gene and protein expression were observed in both oral cancer cell lines. The concentration of 10 μM of norepinephrine significantly inhibited (p ≤ 0.05) migration of SCC-9 and SCC-25 cell lines. Furthermore, there was a significant reduction (p ≤ 0.05) in the effect of norepinephrine on cell migration when the β2-AR was inhibited by propranolol. The blockade by propranolol showed a tendency to reverse the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25.The use of beta-adrenergic receptor agonists could become an adjuvant therapeutic target in the treatment of this malignancy.

Expression of β-adrenergic receptor subtypes in human normal and dilated ureter.

Ureter peristalsis is a basic physiological function regulated by myogenic and neurogenic factors. The distribution and function of β-adrenergic receptors (β-AR) in the human ureter remain unknown. The aim of this study was to investigate the expression of β-AR subtypes in the normal and dilated human ureter. The upper, middle, and lower segments of normal and dilated ureters were collected from patients undergoing surgery for carcinoma of the kidney and upper urinary tract and ureteral stenosis. The mucosa and muscular layers were separated. Expression of β1-AR, β2-AR, and β3-AR mRNA and protein levels were detected by real-time PCR, western blot, and immunohistochemistry. In both mucosa and muscular layers, the mRNA and protein expressions of β1-AR, β2-AR, and β3-AR were lower in the dilated ureter compared with the normal ureter. β1-AR mRNA was significantly decreased (by 76.64%; P<0.01) in the mucosa layer of the middle segment of the dilated ureter. β1-AR and β3-AR mRNA were significantly decreased (by 75.53 and 53.62%, respectively; P<0.01) in the muscular layer of the lower segment of the dilated ureter. Similar findings were observed for protein expression. The downregulation of β-ARs after ureter dilation, particularly for β1-AR and β3-AR in the muscular layer, suggests a potential compensatory mechanism involving increased contraction of the ureter to push urine through the obstruction. Thus, β-ARs may be a potential target for treatment of ureter obstruction.

The effects and possible mechanism of β2AR gene expression in cardiocytes of canines with heart failure.

The objective of the present study was to observe the changes of β2-adrenergic receptor (β2AR) protein expression in a canine model of heart failure (HF), and the function of cardiocytes after transfection with Adv-β2AR. The canine model of chronic HF was induced by rapid right ventricular pacing and cardiocytes were isolated with collagenase II. Cardiocytes were transfected with Adv-β2AR to observe contractile function with a motion edge-detection system of single cells. Expression of β2AR protein in cardiocytes was measured by immunoblotting and the levels of intracellular cAMP were measured by ELISA. Compared with the control group (the sham group), the expression of β2AR protein in HF cardiocytes did not change, but the basal (1 mM Ca2+) contraction amplitude percentage (1.809±0.922 vs. 1.120±0.432%, P<0.05), the maximum contraction amplitude percentage (14.855±2.377 vs. 10.784±2.675%, P<0.01) and the basal levels of intracellular cAMP (9.39±2.54 vs. 5.26±0.95 pmol/ml, n=6, P<0.05) of HF cardiocytes were significantly decreased. However, when HF cardiocytes were transfected with Adv-β2AR and cultured for 48 h, compared with the non-transfected group, the basal contraction amplitude percentage (0.851±0.324 vs. 1.629±0.522%, P<0.05), the maximum contraction amplitude percentage (9.260±2.208% vs. 12.205±1.437%, P<0.01) and the basal levels of intracellular cAMP (5.26±0.95 vs. 9.03±1.03 pmol/ml, n=6, P<0.05) of cardiocytes in the transfected group were significantly increased. In conclusion, the expression of β2AR protein in HF cardiocytes did not change, but contraction function was impaired. The moderate overexpression of β2AR gene in the HF cardiocytes increased the levels of intracellular cAMP and improved contraction function.

Suppression of microRNA-16 protects against acute myocardial infarction by reversing beta2-adrenergic receptor down-regulation in rats.

microRNA-16 (miR-16) has been shown to be up-regulated in ischemic heart. Beta2-adrenoreceptor (β2-AR) exerts cardioprotective property in ischemic injury. This study aims to determine the effect of miR-16 in cardiac injury in rats and the possible involvement of β2-AR in this process. Acute myocardial infarction (AMI) model in rats was induced by ligation of left coronary artery. Neonatal rat ventricular cells (NRVCs) were cultured in vitro tests. The cardiomyocyte model of oxidative injury was mimicked by hydrogen peroxide. The expression of miR-16 was obviously up-regulated and β2-AR was remarkably down-regulated in both AMI rats and NRVCs under oxidative stress. miR-16 over-expression in NRVCs reduced cell viability and increased apoptosis. Conversely, inhibition of endogenous miR-16 with its specific inhibitor reversed these changes. Over-expression of miR-16 using an miR-16 lentivirus in AMI rats markedly increased cardiac infarct area, lactate dehydrogenase and creatine kinase activity, and exacerbated cardiac dysfunction. Lentivirus-mediated knockdown of miR-16 alleviated acute cardiac injury. Moreover, miR-16 over-expression significantly suppressed β2-AR protein expression in both cultured NRVCs and AMI rats, while inhibition of miR-16 displayed opposite effect on β2-AR protein expression. Luciferase assay confirmed that miR-16 could directly target the 3′untranslated region of β2-AR mRNA. miR-16 is detrimental to the infarct heart and suppression of miR-16 protects rat hearts from ischemic injury via up-regulating of β2-AR by binding to the 3′untranslated region of β2-AR gene. This study indicates that targeting miR-16/β2-AR axis may be a promising strategy for ischemic heart disease.

β-Adrenergic receptor gene expression in HIV-associated neurocognitive impairment and encephalitis: implications for MOR-1K subcellular localization.

We previously reported that mRNA expression of the unique alternatively spliced OPRM1 isoform μ-opioid receptor-1K (MOR-1K), which exhibits excitatory cellular signaling, is elevated in HIV-infected individuals with combined neurocognitive impairment (NCI) and HIV encephalitis (HIVE). It has recently been shown that the β2-adrenergic receptor (β2-AR) chaperones MOR-1K, normally localized intracellularly, to the cell surface. Here, we found mRNA expression of the adrenoceptor beta 2 (ADRB2) gene is also elevated in NCI-HIVE individuals, as well as that β2-AR protein expression is elevated in HIV-1-infected primary human astrocytes treated with morphine, and discuss the implications for MOR-1K subcellular localization in this condition.

Effect of adenovirus mediated β2-AR overexpression on IL-10 level secreted by cardiomyocytes of heart failure rats.

The effect of β2-adrenergic receptor (AR) overexpression on interleukin (IL)-10 content secreted by cardiomyocytes of heart failure (HF) rats was investigated. A rat model of chronic HF was established by partially banding abdominal aorta and the cardiomyocytes were isolated with collagenase II. The cardiomyocytes were then transfected with adenovirus type 5-ADRβ2-enhanced green fluorescent protein (EGFP) for 48 h to observe the changes of β2-AR protein expression using western blot analysis. The IL-10 level was detected by ELISA. The experiment was divided into seven groups: Control, HF, HF+EGFP, HF+β2, sham, sham+EGFP and sham+β2 groups. Compared with the sham-operated group, left ventricular diastolic dimension, and left ventricular systolic dimension were increased (P<0.05), whereas ejection fraction and fractional shortening were decreased (P<0.05) in the HF group. Compared with the sham group, the cardiomyocyte survival rate of the HF group was significantly reduced (P<0.05). Compared with the control or sham group, the β2-AR protein level of the HF group showed no significant differences (P>0.05). Compared with the HF and HF+EGFP groups, the expression of β2-AR protein of cardiomyocytes was increased in the HF+β2 group (P<0.05). Compared with the sham group, IL-10 content secreted by cardiomyocytes in the HF group was increased (P<0.05). Compared with the HF and HF+EGFP groups, IL-10 content in the HF+β2 group was increased significantly (P<0.05). In conclusion, the concentration of IL-10 secreted by cardiomyocytes of HF rats was increased. The overexpression of β2-AR in the cardiomyocytes of HF rats was able to enhance the secretion of IL-10.

Abstract 288: Rnd3/RhoE Regulates Cardiac Ryanodine Receptor Type 2 Stability

Rationale: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration and proliferation. The biological function of Rnd3 in the heart remains unexplored. Objective: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism. Methods and Results: By loss-of-function approaches, we discovered a new role in which Rnd3 stabilizes the ryanodine receptor type 2 (RyR2) Ca 2+ release channel. Genetic deletion of Rnd3 in mice resulted in embryonic lethality with heart failure and arrhythmia. Both Rnd3 -/- embryonic and Rnd3 +/- adult cardiomyocytes showed severe Ca 2+ leakage. Single channel assessment showed the destabilized RyR2 channel, and this irregular spontaneous Ca 2+ release was curtailed by protein kinase A (PKA) inhibitor treatment. Further studies found that RyR2 protein was hyperphosphorylated by PKA in the mutant heart. Remarkable increases in the PKA activity along with elevated cyclic adenosine monophosphate levels were detected in vivo in Rnd3-null embryos and in vitro in neonatal rat cardiomyocytes and non-cardiac cell lines with Rnd3 knockdown. Moreover, we found increasing β 2 -adrenergic receptor (β 2 AR) protein levels, but no correlated mRNA changes in both the Rnd3-null heart and non-cardiac cells with Rnd3 knockdown. Immunoprecipitation analysis demonstrated that Rnd3 and β 2 AR physically interacted. Multiple post-translational modification analyses of β 2 AR revealed that downregulation of Rnd3 attenuated β 2 AR protein lysosomal targeting and ubiquitination, which in turn resulted in the elevation of β 2 AR protein levels contributing to the activation of PKA signaling. Rnd3 deficiency had no effects on the hydroxylation- and sumoylation-mediated β 2 AR protein degradation. Conclusion: Rnd3 is a unique stabilizer of RyR2 that impacts intracellular Ca 2+ handling in the heart.

Agonist-induced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o− cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Crosstalk between adrenergic and toll-like receptors in human mesenchymal stem cells and keratinocytes: a recipe for impaired wound healing.

Previous studies demonstrate that skin wounds generate epinephrine (EPI) that can activate local adrenergic receptors (ARs), impairing healing. Bacterially derived activators of Toll-like receptors (TLRs) within the wound initiate inflammatory responses and can also impair healing. In this study, we examined the hypothesis that these two pathways crosstalk to one another, using EPI and macrophage-activating lipopeptide-2 (MALP2) to activate ARs and TLR2, respectively, in human bone marrow-derived mesenchymal stem cells (BM-MSCs) and neonatal keratinocytes (NHKs). BM-MSCs exposed to EPI significantly (p < .05) increased TLR2 message (sevenfold BM-MSCs), TLR2 protein (twofold), and myeloid differentiation factor 88 (MyD88) (fourfold). Conversely, activation of TLR2 by MALP2 in these cells increased β2-AR message (twofold in BM-MSCs, 2.7-fold in NHKs), β2-AR protein (2.5-fold), phosphorylation of β-AR-activated kinase (p-BARK, twofold), and induced release of EPI from both cell types (twofold). Treating cells with EPI and MALP2 together, as would be encountered in a wound, increased β2-AR and p-BARK protein expression (sixfold), impaired cell migration (BM-MSCs- 21%↓ and NHKs- 60%↓, p < .002), and resulted in a 10-fold (BM-MSCs) and 51-fold (NHKs) increase in release of IL-6 (p < .001) responses that were remarkably reduced by pretreatment with β2-AR antagonists. In vivo, EPI-stressed animals exhibited impaired healing, with elevated levels of TLR2, MyD88, and IL-6 in the wounds (p < .05) relative to nonstressed controls. Thus, our data describe a recipe for decreasing cell migration and exacerbating inflammation via novel crosstalk between the adrenergic and Toll-like receptor pathways in BM-MSCs and NHKs.