Adrenergic Receptor Desensitization Research Articles

Long-term Xenopus laevis tadpole -heart-organ-culture: Physiological changes in cholinergic and adrenergic sensitivities of tadpole heart with thyroxine-treatment

The present study clarified changes in physiological sensitivities of cultured Nieuwkoop and Faber stage 57 Xenopus laevis tadpole-organ-heart exposed to thyroxine (T4) using acetylcholine (ACh), norepinephrine (NE) and atropine. For preliminary life span and the chemical tests, 60% minimum essential medium (MEM), two types of modified Hank's balanced salt-solution-culture-media (MHBSS-CM) I and II containing relatively lower concentrations of amino acids and collagen were prepared. In preliminary lifespan-test of cultured tadpole hearts, the hearts maintained in 60% MEM was 50 days on average, whereas that of the tadpole-hearts in MHBSS-CMs was extended by 109 days on average, showing superior effectiveness of MHBSS-CMs. 4 min-stimulation by 5 × 10−9 M T4 tended to increase the tadpole heartbeat. 10−9 M ACh decreased the tadpole heartbeat. Frog-heart at 2–4 weeks after metamorphosis completion and tadpole heart treated with 5 × 10−10 M T4 for 45 h also responded to 10−9 M ACh, and low-resting hearts were restored to the control level with the competitive muscarinic antagonist 10−8 M atropine, whereas excessive exposure of 10−5 M atropine to T4-treated tadpole heart did not increase heartbeat in spite of the increased frog heartbeat over the control. 10−14 —10−12 M NE increase the tadpole heartbeat in a concentration-dependent manner, however, 10−12 M NE did not act to stimulate adrenergic receptors on both T4-treated tadpole- and the frog-hearts. These results suggest that T4 induces the desensitization of atropine-sensitive muscarinic and adrenergic receptors in organ-cultured tadpole-heart.

PSAT015 A Rare Case of Incidental Massive Pheochromocytoma with Ectopic Parathyroid Hormone Secretion

Abstract Background Pheochromocytoma (PC) is a rare neuroendocrine tumor with an annual incidence of 2 to 8 cases per million that can secrete excess catecholamines precipitating hyper-adrenergic symptoms. In rarer instances, PCs can also secrete substances other than catecholamines. Case A 51-year-old female underwent MRI lumbar spine that demonstrated an incidental 13 cm right adrenal mass. History was significant for newly diagnosed hypertension and a five-year history of episodic headaches, diaphoresis, palpitations and anxiety. A previous cardiac evaluation including Holter monitor, echocardiogram, and stress test was unrevealing. Physical exam was significant only for chronic back pain and BMI 20.4. Labs revealed elevated serum catecholamines with metanephrines and normetanephrines >40x and >65x the upper limit of normal respectively, elevated urine catecholamines and elevated chromogranin A suggestive of PC. Labs also demonstrated mild hypercalcemia >10.3 mg/dL (8.6-10.4 mg/dL) with elevated PTH 78 pg/mL (14-64 pg/mL) and low vitamin D 25-OH 25 ng/mL (30-100 ng/mL). Calcitonin, albumin and phosphorus were normal. She was treated with non-competitive alpha and beta-blockade and metyrosine before undergoing successful right adrenalectomy. Post-operative labs demonstrated normalization of catecholamines, PTH and calcium. Surgical pathology confirmed the diagnosis of PC and demonstrated tumor cells which stained for PTH confirming ectopic PTH secretion, a rare phenomenon. The PC was very large with an elevated PASS (Pheochromocytoma of the Adrenal Gland Scaled Score) indicating increased risk for malignancy/recurrence; however, there was low Ki-67 activity and no capsular involvement, which suggested benign behavior. Furthermore, an MIBG scan was negative for metastatic disease, and an extensive genetic workup for familial PC/PGL (including MEN, RET, SDHx) was negative. Conclusion PC is a rare neuroendocrine tumor that can secrete excess catecholamines precipitating hyper-adrenergic symptoms and potentially fatal complications including hypertensive crises, cardiovascular disease, ischemia and multi-organ failure if untreated. Consequently, early recognition of PCs is imperative. The patient in this case had a five-year history of symptoms seen with PC and newly diagnosed hypertension despite normal BMI and negative cardiac workup. However, she had no evaluation for secondary causes of hypertension. Despite the large tumor size and significantly elevated catecholamines, she functioned without significant morbidity potentially due to adrenergic receptor desensitization. This case also highlights the rare phenomenon of ectopic secretion in PCs, specifically ectopic PTH secretion. A 2019 systemic review identified only 150 cases of pheochromocytomas/paragangliomas with ectopic hormone secretion – of those, only 17 secreted PTH or PTH-related peptide [1]. It is important to consider ectopic secretion in patients with PCs as they may have additional clinical manifestations associated with the hormone or substance secreted. Fortunately, this patient had no overt manifestations of hyperparathyroidism or hypercalcemia.

Vasoplegic Syndrome after Cardiopulmonary Bypass in Cardiovascular Surgery: Pathophysiology and Management in Critical Care.

Vasoplegic syndrome (VS) is a common complication following cardiovascular surgery with cardiopulmonary bypass (CPB), and its incidence varies from 5 to 44%. It is defined as a distributive form of shock due to a significant drop in vascular resistance after CPB. Risk factors of VS include heart failure with low ejection fraction, renal failure, pre-operative use of angiotensin-converting enzyme inhibitors, prolonged aortic cross-clamp and left ventricular assist device surgery. The pathophysiology of VS after CPB is multi-factorial. Surgical trauma, exposure to the elements of the CPB circuit and ischemia-reperfusion promote a systemic inflammatory response with the release of cytokines (IL-1β, IL-6, IL-8, and TNF-α) with vasodilating properties, both direct and indirect through the expression of inducible nitric oxide (NO) synthase. The resulting increase in NO production fosters a decrease in vascular resistance and a reduced responsiveness to vasopressor agents. Further mechanisms of vasodilation include the lowering of plasma vasopressin, the desensitization of adrenergic receptors, and the activation of ATP-dependent potassium (KATP) channels. Patients developing VS experience more complications and have increased mortality. Management includes primarily fluid resuscitation and conventional vasopressors (catecholamines and vasopressin), while alternative vasopressors (angiotensin 2, methylene blue, hydroxocobalamin) and anti-inflammatory strategies (corticosteroids) may be used as a rescue therapy in deteriorating patients, albeit with insufficient evidence to provide any strong recommendation. In this review, we present an update of the pathophysiological mechanisms of vasoplegic syndrome complicating CPB and discuss available therapeutic options.

Morphological manifestations of the dynamics of catecholamines binding by erythrocytes during activation and blockade of adrenergic regulatory mechanisms

Objective - to study, the dynamics of catecholamine binding on erythrocytes when modeling the stimulation and blockade of adrenergic regulation mechanisms using the cytological method
 Material and methods. The number of catecholamine granules on erythrocytes was determined using silver nitrate impregnation under conditions of administration of anapriline -adrenergic receptor blocker (2 mg/kg), acute stress, activation of noradrenergic systems (maprotiline, 10 mg/kg) and a combination of these effects.
 Results. The number of catecholamine granules in intact animals is 145-155 pieces/40 erythrocytes. Medium-sized granules are more common (0.6-0.9 m). After the administration of a -adrenergic receptor blocker, the total number of catecholamine granules decreases 2.8 times due to large and medium-sized granules. Under conditions of acute stress, the total number of granules increases almost 2 times due to small granules, which may be a sign of sensitization of erythrocyte membranes to catecholamines. Stimulation of the noradrenergic system causes a 20% decrease in the number of catecholamine granules due to a decrease in the number of small and medium sized granules. Under stress against the background of activation of the noradrenergic system, the number of granules on erythrocytes is reduced, which may be a sign of adrenergic receptor desensitization.
 Conclusions. The number of catecholamine granules on erythrocytes decreases after the administration of a -adrenergic receptor blocker and increases during acute stress. Stimulation of the noradrenergic system is accompanied by a decrease in the binding of catecholamines, especially under conditions of acute stress, which indicates desensitization of erythrocyte adrenergic receptors. The cytological method is sensitive enough to observe the reception of catecholamines by erythrocytes when exposed to adrenergic structures.

Epinephrine evokes shortening of human airway smooth muscle cells following β2 adrenergic receptor desensitization.

Epinephrine (EPI), an endogenous catecholamine involved in the body’s fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiological functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating β2-adrenergic receptors (β2ARs). Conventionally, the selective β2AR agonism of EPI was thought to be, in part, due to a predominance of β2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous “switch-like” activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of β2ARs and/or under experimental conditions that induce β2AR tachyphylaxis. EPI-induced procontractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or β2-agonist insensitivity in patients with difficult-to-control, disease subtypes.

Abstract 10247: Establishing a Therapeutically Modifiable ROS/RNS-Beta 1 Adrenergic Receptor Desensitization and Internalization Axis in Aging Coronary Microvessels

Introduction: Coronary Microvascular Disease (CMD) causes hyperconstriction, reduced perfusion, and chronic angina in aging. We previously showed increased coronary microvascular ROS and reduced NO/glutathione in aging rats correlating with reduced β1 Adrenergic receptor (β1ADR) dilation. Adipose Stromal Vascular Fraction (SVF) therapy reverses oxidative stress in CMD to improve β1ADR function. Our objective is to elucidate a mechanism for SVF’s recovery of β1ADR function in pursuit of a cell therapy to treat CMD. Hypothesis: β1ADR dilation is reduced in aging due to ROS-mediated receptor desensitization & internalization, reversible by inhibition of GRK2/dynamin & SVF antioxidation. Methods: Rat coronary microvessels were isolated from young (YC, 3-6 months), old (OC, 24 months), and old + SVF injection (OSVF, 24 months). RNAseq (n = 3 per group) and immunofluorescence (n ≥ 5 per group) were used to analyze pro/antioxidant gene & protein expression. Using pressure myography, dilation to isoproterenol was evaluated at baseline and with ROS (hydrogen peroxide and menadione) or RNS (sodium nitroprusside, SNP) +/- inhibitors of receptor desensitization (GRK2 via paroxetine) and internalization (dynamin via dynasore). Significance was set at p < .05 utilizing Two-Way Repeated Measures ANOVA with Holm-Sidak post-hoc analysis. Results: Aging (n = 16) reduced dilation to isoproterenol, reversed to youthful (n = 17) levels by SVF (n = 16). In YC/OSVF, ROS (n = 10 & 6) attenuated dilation reversed by dynasore (n = 8) and paroxetine (n = 7). In OC, SNP (n = 7), dynasore (n = 10), and paroxetine (n = 5) restored dilation. Aging increased prooxidant genetic (xanthine oxidase) and protein (NOX4) expression and increased antioxidant genetic (catalase) but not protein expression. SVF reduced prooxidant genetic (dynamin related protein 1) and protein (NOX4) expression with increased antioxidant genetic (glutathione synthetase) and protein (SOD2) expression. Conclusion: ROS in aging causes β1ADR dysfunction via desensitization & internalization, as their inhibition preserves β1ADR function. We name this interaction the ROS/RNS- β1ADR Desensitization & Internalization Axis , which was therapeutically modulated by SVF antioxidation to restore β1ADR function.

Single-cell Analysis of β2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy.

G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization. To develop a quantitative fluorescence Microscopy assay to study β2- Adrenergic receptor expression and desensitization. β2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of β2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software. The method is sensitive and allows for the simultaneous measurement of surface and total expression of β2-AR. A highly accurate method is described for measuring β2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of β2-Adrenergic receptor and can be applied to essentially any other GPCR.

Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle.

Key points Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity.Although whole‐body glucose clearance is normal, 1‐month‐old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation.Impaired glucose‐stimulated insulin secretion in IUGR lambs is due to lower intra‐islet insulin availability and not from glucose sensing.We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole‐body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies.IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose‐stimulated insulin secretion and insulin‐stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin‐sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose‐stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR‐AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole‐body GUR were not different from controls. Of importance, ADRβ2 stimulation with β1/β3 inhibition increases both insulin sensitivity and whole‐body glucose utilization in IUGR lambs. In IUGR and IUGR‐AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR‐AR skeletal muscle than in controls but GLUT1 was greater in IUGR‐AR. ADRβ2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR‐AR lambs heart rates were greater, which was independent of cardiac ADRβ1 activation. We conclude that targeted ADRβ2 stimulation improved whole‐body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch‐up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

EP2 and EP4 receptor antagonists: Impact on cytokine production and β2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for β2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) β2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores β2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Human a1A‐adrenergic receptor phosphorylation, internalization and desensitization.

The a1A‐adrenergic receptor (a1A‐AR) is a G protein coupled‐receptor (GPCR) that signals through the Gq/ phospholipase C/ calcium release pathway. This adrenergic receptor subtype modulates important cellular responses and it is also involved in the pathogenesis of diseases. Phosphorylation of intracellular loops and the carboxyl terminal tail of a1A‐AR is part of their fine regulation, generally associated with desensitization/internalization processes, in which different protein kinases participate, Including second messenger‐activated protein kinases, such as protein kinase C isoforms (PKCs) and G protein‐coupled receptor kinases (GRKs).In silico analysis of the human a1A‐AR amino acid sequenced, suggested a series of possible phosphorylation sites targeted by PKCs, GRKs and also for the glycogen synthase protein kinase‐3 (GSK3). GSK3 is an important element into signal transduction of diverse receptor systems, and the phosphorylation on specific substrates can modulates enzyme activity, subcellular localization, stability of the target protein and protein‐protein interactions. We observed using a1A‐AR immunopurification and mass spectrometry analysis that in cells treated with noradrenaline, oxymetazoline or phorbol esters four serines that are target of GSK3: S229, S258, S352 and S381, are phosphorylated. However, the specific GSK3 inhibitor, SB216763, did not block the a1A‐AR desensitization induced by noradrenaline or oxymetazoline. This suggested that other protein kinases play major roles in such agonist‐induced desensitization. Interestingly, phorbol ester‐induced a1A‐AR desensitization was increased by the GSK3 inhibitor. In cellulo colocalization assays of a1A‐AR‐GSK3, showed increased interaction of these proteins in the presence of agonists and phorbol esters. Interestingly, expression of a GSK3 dominant‐negative mutant interferes with a1A‐AR internalization in response to NA and OXY, but this effect does not occur when cells are pretreated with PMA. All these data suggest the possibility that GSK3 might play a role in the regulation of a1A‐AR function and localization.Partially supported by Grants from Dirección General de Asuntos del Personal Académico (IN200718) and Consejo Nacional de Ciencia y Tecnología (Fronteras 882 and 253156).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tips for management of arrhythmias in endocrine disorders from an European Heart Rhythm Association position paper.

In endocrine diseases, hormonal changes, electrolyte abnormalities, and the deterioration of heart structure can lead to various arrhythmias. In diabetic patients, hypoglycemia, hyperglycemia, and hypokalemia can trigger arrhythmias, and diabetic cardiomyopathy can also cause electrical and structural remodeling to form substrates for arrhythmias. The risk of atrial fibrillation (AF) increases in hyperthyroidism; however, the prevalence of ventricular arrhythmias in hypothyroidism is higher. Besides AF and ventricular tachycardias, bradycardias and atrioventricular blocks can also be seen in pheochromocytoma due to the desensitization of adrenergic cardiovascular receptors. The correction of metabolic and electrolyte disturbances in patients with adrenal cortex disease should be the main approach in the prevention and treatment of arrhythmias. Early initiation of treatment in patients with acromegaly seems to decrease the development of cardiac remodeling and ventricular arrhythmia. Early and late after depolarizations due to hypercalcemia in hyperparathyroidism can lead to life-threatening ventricular arrhythmias. This elegant position paper provides important recommendations regarding prevention and treatment of arrhythmias for specific endocrine disorders.

Abstract 469: Targeting Beta Adrenergic Receptor Resensitization Aids in Preservation of Receptor Function

Beta adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and is an integral contributor to heart failure. We have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also generated a small molecule from our in silico predictions that could target the interface of I2PP2A and PP2A binding to find that disruption of the PP2A/I2PP2A interaction underlies receptor function. We will use this small molecule to look at preservation of βAR function and amelioration of cardiac function. To test whether resensitization is altered in heart failure we used plasma membrane and endosomal fractions from non-failing and paired pre- and post-LVAD samples to show PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase activity as a measure of recovery in βAR function. Our studies showed that endosomal fractions from human heart failure samples had elevated PI3K activity associated with reduced PP2A activity supporting the idea that βAR resensitization is inhibited in human heart failure samples. Since human heart failure samples have inhibited resensitization we tested the underlying mechanisms regulating βAR resensitization. Thus ongoing studies suggest that targeting the resensitization of βAR could provide beneficial cardiac remodeling in conditions of chronic mechanical overload and will be further discussed.

The Beta 1 and Beta 2 Adrenergic Receptors Exhibit Differential Coupling to the Cyclic AMP Sensor NCS‐Rapgef2

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. Both of these receptors couple to Gsα. Their activation causes cAMP elevation, which in turn controls cellular signaling through its downstream effectors PKA and the cAMP guanine nucleotide exchange factors (GEFs) Epac1 and Epac2. The β2AR has also been shown to signal via engagement of β‐arrestin, which acts as a scaffold for a variety of signaling proteins, including the MAP kinase ERK. We have recently characterized a neuronal and endocrine‐specific cAMP sensor, a GEF related to Epac 1 and 2, and to the previously characterized non‐cAMP‐activated GEF PDZ‐GEF1, which we have named NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). This sensor mediates activation of ERK leading to neuritogenesis in the PC12 and NS‐1 neuroendocrine cell lines (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014). We have created NS‐1 cell lines stably expressing either β1AR or β2AR, and examined signaling to each of the three cAMP sensors present in these cells following treatment with isoproterenol. In β1AR‐expressing cells, agonist treatment caused activation of all measurable cAMP‐dependent signaling pathways in these cells: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2/ERK‐dependent neuritogenesis. In contrast, agonist stimulation of β2AR‐expressing cells caused isoproterenol‐initiated Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but did not couple to NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, a biosensor that allowed for continuous real‐time cyclic AMP measurements was co‐expressed in each cell line (Emery et al., Peptides, 79: 39, 2016). In β2AR‐expressing cells, the maximal effect of isoproterenol on cAMP was observed after 10 minutes of treatment and decreased rapidly thereafter. In contrast, isoproterenol‐dependent β1AR activation caused persistent cAMP elevation, observed at approximately maximal levels at least 40 minutes following agonist addition. Unlike the mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent), ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) most likely occurs in a cellular compartment restricted from transcriptional regulation, which is required for neuritogenesis in this cell type (Ravni et al., Mol. Pharmacol. 73: 1688, 2008). We conclude that there is an inverse relationship between adrenergic receptor desensitization, and engagement of NCS‐Rapgef2 of sufficient duration to support the sustained activation of ERK necessary to promote neuritogenesis in NS‐1 cells. We notice the same inverse relationship in receptors for dopamine (D1) and adenosine (A2A), as well as the neuropeptides PACAP, VIP, and GLP‐1 (PAC1, VPAC1, VPAC2, and GLP‐1R).Support or Funding InformationThis work was supported by NIMH Intramural Research Program Project ZIAMH002386 and by a 2014 NARSAD Young Investigator Grant to A.C.E. from the Brain and Behavior Research Foundation (Grant 21356).

Effect of Sphingosine 1-Phosphate on Cyclo-Oxygenase-2 Expression, Prostaglandin E2 Secretion, and β2-Adrenergic Receptor Desensitization.

Tachyphylaxis of the β2-adrenergic receptor limits the efficacy of bronchodilatory β2-agonists in respiratory disease. Cellular studies in airway smooth muscle (ASM) have shown that inflammatory mediators and infectious stimuli reduce β2-adrenergic responsiveness in a cyclo-oxygenase (COX)-2-mediated, prostaglandin E2 (PGE2)-dependant manner. Herein, we show that sphingosine 1-phosphate (S1P), a bioactive sphingolipid that plays an important role in pathophysiology of asthma, also induces β2-adrenergic receptor desensitization in bronchial ASM cells and exerts hyporesponsiveness to β2-agonists. We treated ASM cells with S1P (1 μM) for up to 24 hours and then examined the temporal kinetics of COX-2 mRNA expression, protein up-regulation, and PGE2 secretion. S1P significantly enhanced COX-2 expression and PGE2 secretion, and this was repressed by the selective COX-2 inhibitor celecoxib, the corticosteroid dexamethasone, or small interfering RNA (siRNA) knockdown of COX-2 expression. In combination with another proinflammatory mediator found elevated in asthmatic airways, the cytokine TNF-α, we observed that S1P-induced COX-2 mRNA expression and protein up-regulation and PGE2 secretion from ASM cells were significantly enhanced. Notably, S1P induced heterologous β2-adrenergic desensitization, as measured by inhibition of cyclic adenosine monophosphate production in response to the short-acting β2-agonist, salbutamol, and the long-acting β2-agonist, formoterol. Taken together, these data indicate that S1P represses β2-adrenergic activity in ASM cells by increasing COX-2-mediated PGE2 production, and suggest that this bioactive sphingolipid found elevated in asthma may contribute to β2-adrenergic desensitization.

IL-17A increases TNF-α-induced COX-2 protein stability and augments PGE2 secretion from airway smooth muscle cells: impact on β2 -adrenergic receptor desensitization.

IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2 ) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 induces beta-2 adrenergic receptor desensitization in β2AR desensitization mice model.

It is well-known that chronic administration of β2AR agonists can induce β2AR desensitization. Our previous study showed that Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) overexpression induced beta-2 adrenergic receptor (β2AR) desensitization in airway smooth muscle cells. The purpose of this study was to further study the function of RhoGDI2 in β2AR desensitization by β2AR desensitization mouse model. Studies were performed using a β2AR desensitization mice model induced by salbutamol. The mice were randomly divided into five groups (n=45): RhoGDI2 overexpression group (n=10); RhoGDI2 siRNA group (n=10); empty viral vector group (n=10); experimental control group (n=10); blank control group-without any drug treatment (n=5). The first four groups were used the same methods and the same dose to establish β2AR desensitization mice model by salbutamol. The first three groups that salbutamol-treated were used for intratracheal delivery of lentiviral vectors. Airway hyperreactivity was measured through a whole-body plethysmograph system. RhoGDI2, β2AR, GRK2 mRNA and protein expression levels were then detected by RT-PCR and western blot analyses in fresh lung tissues. As well as the activity of GRK was assessed by light-dependent phosphorylation of rhodopsin. We successfully constructed β2AR desensitization mouse model. As expected, airway responsiveness after inhaling acetylcholine chloride (Ach) was markedly increased in the RhoGDI2 overexpression group compared to experimental control group and blank control group when concentrations of Ach was 45 mg/mL (all P<0.05), while, it was markedly decreased in the RhoGDI2 siRNA group compared to experimental control group (P<0.05). RhoGDI2, GRK2 expressions and GRK enzymatic activity were significantly increased in RhoGDI2 overexpression group compared to experimental control group and blank control group (all P<0.05). RhoGDI2, GRK2 expressions and GRK enzymatic activity were significantly decreased in RhoGDI2 siRNA group compared to experimental control group and blank control group (all P<0.05). Conversely, β2AR expression were significantly lower in RhoGDI2 overexpression group compared to experimental control group and blank control group (all P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. To sum up, our present studies found that RhoGDI2 might induce β2AR desensitization and GRK2 might take part in RhoGDI2-mediated β2AR desensitization.

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells.

Beta-2 adrenergic receptor (β2AR) downregulation is critical to asthma rescue therapy; however, tolerance, also known as β2AR or bronchodilator desensitization, mechanisms potentially resulting in life-threatening rescue treatment failure remain poorly understood. Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) gene from ASMCs was amplified by RT-PCR, and RhoGDI2 gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI2 expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI2, β2AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β2AR was observed by radioligand receptor binding assay in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. RhoGDI2 vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI2, GEF and G-protein-coupled receptor kinase 2 (GRK2) expressions significantly increased in the RhoGDI2 overexpression group compared to control and negative control groups (all P<0.05). Conversely, β2AR and GAP expressions were significantly lower in the RhoGDI2 overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. Control and negative control groups exhibiting β2AR density more than 2-fold higher than that observed in the RhoGDI2 overexpression group. RhoGDI2 reduces β2AR density, potentially by reducing β2AR and GAP expressions and increase GEF and GRK2 expressions. Thus, RhoGDI2 is central in cellular β2AR desensitization, though this full mechanism and intermediates merit further investigation.

β‐adrenergic receptor agonists known to stimulate β‐arrestin‐dependent signaling inhibit CFTR activity, cell migration and airway epithelial restitution

Previously, the β‐adrenergic receptor antagonist carvedilol was shown to function as an inverse agonist, capable of stabilizing a receptor conformation uncoupled from Gs, but able to stimulate β‐arrestin‐dependent signaling and induce desensitization. In this study, measurements of cell migration using impedance sensing technology showed that stimulation of normal human bronchial epithelial cells and Calu‐3 cells with epinephrine or salbutamol reduced cell migration and increased the time required for epithelial restitution. Pretreatment with CFTR inhibitor CFTRinh‐172 or CFTR silencing blocked the effect of epinephrine and salbutamol. Carvedilol also inhibited migration and restitution to the same extent as epinephrine and salbutamol. Moreover, short circuit current (Isc) measurements revealed that unlike β‐agonists which increased Isc, carvedilol decreased basal current and the peak current following 8‐cpt cAMP stimulation. Pretreatment with carvedilol also abolished the effect of 8‐cpt cAMP on Cl− secretion. The carvedilol effect was abolished by pretreatment with the β2‐adrenergic receptor antagonist ICI‐118551, which also rescued Clsecretion following 8‐cpt cAMP stimulation. Therefore, agonists that activate β‐arrestin signaling and facilitate β2 adrenergic receptor desensitization inhibit cell migration and wound repair by a mechanism involving CFTR inhibition.

Differential TNF production by monocyte subsets under physical stress: Blunted mobilization of proinflammatory monocytes in prehypertensive individuals

Elevated blood pressure (BP) and infiltration of the vasculature by monocytes contribute to vascular pathology; but, monocyte migratory characteristics based on differing inflammatory potential under adrenergic activation remains unclear. We compared nonclassical (CD14(+)CD16(++); HLA-DR(+)), intermediate (CD14(++)CD16(+); HLA-DR(++)), and classical (CD14(++)CD16(-); HLA-DR(+/-)) monocyte trafficking and their LPS-stimulated TNF production in response to a physical stressor (20-min treadmill exercise at 65-70% VO(2peak)) in participants with high prehypertension (PHT), mild PHT or normal BP (NBP). To determine adrenergic receptor (AR) sensitivity, pre-exercise cells were also treated with isoproterenol (Iso). When cells were stimulated with LPS, the CD16 molecules were downregulated, and monocyte subsets were differentiated based on HLA-DR expression. Monocyte subpopulations (as % of total monocytes) and intracellular TNF production were evaluated by flow cytometry. TNF production in all subsets decreased post-exercise and with ex-vivo incubation with Iso, irrespective of BP (p<0.001), with nonclassical and intermediate monocytes being a major source of TNF production. Overall, % nonclassical monocytes increased, % intermediate did not change, whereas % classical decreased post-exercise (p<0.001). However, % increase in nonclassical monocytes under exercise-induced adrenergic activation was blunted in high PHT individuals (p<0.05), but not in individuals with mild PHT and NBP. These findings extend our previous reports by showing that the mobilization of proinflammatory monocytes under physical stress is attenuated with even mild BP elevation. This may be indicative of monocytic AR desensitization and/or greater adhesion of "proinflammatory" monocytes to the vascular endothelium in hypertension with potential clinical implications of vascular pathology.

47. Differing trafficking and TNF-alpha production by monocyte subsets under adrenergic activation: Blunted mobilization of “pro-inflammatory” monocytes in individuals with elevated blood pressure

Elevated blood pressure (BP) and infiltration of the vasculature by monocytes contribute to vascular pathology; but, monocyte migratory characteristics based on differing inflammatory potential under adrenergic activation remains unclear. We compared “non-classical” (CD16++CD14+), “intermediate” (CD16+CD14++), and “classical” (CD16-CD14++) monocyte trafficking and their LPS-stimulated TNF-alpha production in response to a physical stressor (20-min treadmill exercise at 65–70% VO2peak) in participants ( n = 77) with high pre-hypertension (PHT), mild PHT or normal BP (NBP). To determine adrenergic receptor sensitivity, pre-exercise cells were also treated with isoproterenol (Iso) before LPS stimulation. Monocyte subpopulations and intracellular TNF-alpha production were evaluated by flow cytometry. Epinephrine and norepinephrine levels were significantly elevated during exercise. Overall, % non-classical monocytes increased, % intermediate did not change, whereas % classical decreased post-exercise ( p < .001). However, the % increase in non-classical monocytes under adrenergic activation was blunted in PHT individuals ( p < .05). TNF-alpha production in all subsets decreased post-exercise and with in-vitro Iso treatment, irrespective of BP ( p < .001), with nonclassical and intermediate monocytes being a major source of TNF-alpha production. These findings extend our previous reports by showing that the mobilization of “pro-inflammatory” monocytes under sympathoadrenal activation is attenuated with even mild BP elevation. This may be indicative of monocytic adrenergic receptor desensitization and/or greater adhesion of “pro-inflammatory” monocytes to the vascular endothelium in hypertension with clinical implications of vascular pathology.