Beta-Adrenergic Receptor Polymorphism and Patho-Genetics of Trauma: A Transformational Frontier of Personalized Medicine in Neurotrauma.

Trauma is a serious public health issue, and remains a major cause of mortality and disability worldwide. The notion that genetic factors contribute to an individual's response to traumatic injury has advanced significantly. Genetic variations in severely injured patients have been linked to mortality, morbidity, and psychological outcomes. We conducted a comprehensive review of beta-adrenergic receptor polymorphisms and their impact on the pathogenetics of traumatic injuries, which could pave the way for a transformational frontier of personalized medicine in neurotrauma. It remains unclear why some individuals are vulnerable to worse outcomes, whereas others are resilient. Although genetic factors may be significant, the intricate interplay between environmental and genetic factors may be responsible for variations in the presentation and outcome after injury. Recent advancements in genetic analysis and molecular physiology have helped to shed light on the causes of such variability. Although exposure to trauma can initiate a cascade of stress-related responses, these responses alone are insufficient to explain etiopathogenesis. Therefore, gaining insights into how trauma and genetic predispositions to adrenergic variations interact at the molecular level to affect an individual's susceptibility and recuperation could provide an essential understanding of the molecular pathogenesis of traumatic injuries. Therefore, it is imperative to identify potential genetic and physiological markers to guide early management and prognosis of trauma. Such knowledge could pave the way for the discovery of novel biomarkers that can identify a transdiagnostic subgroup that is at high risk and requires early intervention. This could lead to the adoption of personalized medical approaches in neurotrauma care.

CD44 regulates Epac1-mediated ß-adrenergic-receptor-induced Ca2+-handling abnormalities: implication in cardiac arrhythmias.

BACKGROUND: Sustained, chronic activation of ß-adrenergic receptor (ß-AR) signaling leads to cardiac arrhythmias, with exchange proteins directly activated by cAMP (Epac1 and Epac2) as key mediators. This study aimed to evaluate whether CD44, a transmembrane receptor mediating various cellular responses, participates in Epac-dependent arrhythmias. METHODS: The heart tissue from CD44 knockout (CD44-/-) mice, cultured HL-1 myocytes and the tissue of human ventricle were used for western blot, co-immunoprecipitaiton and confocal studies. Line-scanning confocal imaging was used for the study of cellular Ca2+ sparks on myocytes. Optical mapping and intra-cardiac pacing were applied for arrhythmia studies on mice's hearts. RESULTS: In mice, isoproterenol, a ß-AR agonist, upregulated CD44 and Epac1 and increased the association between CD44 and Epac1. Isoproterenol upregulated the expression of phospho-CaMKII (p-CaMKII), phospho-ryanodine receptor (p-RyR), and phospho-phospholamban (p-PLN) in mice and cultured myocytes; these effects were attenuated in CD44-/- mice compared with wild-type controls. In vitro, isoproterenol, 8-CPT-cAMP (an Epac agonist), and osteopontin (a ligand of CD44) significantly upregulated the expression of p-CaMKII, p-RyR, and p-PLN; this effect was attenuated by CD44 small interfering RNA (siRNA). In myocytes, resting Ca2+ sparks were induced by isoproterenol and overexpressed CD44, which were prevented by inhibiting CD44. Ex vivo optical mapping and in vivo intra-cardiac pacing studies showed isoproterenol-induced triggered events and arrhythmias in ventricles were prevented in CD44-/- mice. The inducibility of ventricular arrhythmias (VAs) was attenuated in CD44-/- HF mice compared with wild-type HF controls. In patients, CD44 were upregulated, and the association between CD44 and Epac1 were increased in ventricles with reduced contractility. CONCLUSION: CD44 regulates ß-AR- and Epac1-mediated Ca2+-handling abnormalities and VAs. Inhibition of CD44 is effective in reducing VAs in HF, which is potentially a novel therapeutic target for preventing the arrhythmias and sudden cardiac death in patients with diseased hearts.

Dual-omics reveals temporal differences in acute sympathetic stress-induced cardiac inflammation following α1 and ß-adrenergic receptors activation.

Sympathetic stress is prevalent in cardiovascular diseases. Sympathetic overactivation under strong acute stresses triggers acute cardiovascular events including myocardial infarction (MI), sudden cardiac death, and stress cardiomyopathy. α1-ARs and ß-ARs, two dominant subtypes of adrenergic receptors in the heart, play a significant role in the physiological and pathologic regulation of these processes. However, little is known about the functional similarities and differences between α1- and ß-ARs activated temporal responses in stress-induced cardiac pathology. In this work, we systematically compared the cardiac temporal genome-wide profiles of acute α1-AR and ß-AR activation in the mice model by integrating transcriptome and proteome. We found that α1- and ß-AR activations induced sustained and transient inflammatory gene expression, respectively. Particularly, the overactivation of α1-AR but not ß-AR led to neutrophil infiltration at one day, which was closely associated with the up-regulation of chemokines, activation of NF-κB pathway, and sustained inflammatory response. Furthermore, there are more metabolic disorders under α1-AR overactivation compared with ß-AR overactivation. These findings provide a new therapeutic strategy that, besides using ß-blocker as soon as possible, blocking α1-AR within one day should also be considered in the treatment of acute stress-associated cardiovascular diseases.

Radioligand Binding to Quantify Adrenergic Receptor Expression in the Heart.

ß-adrenergic receptors regulate cardiac function in both the healthy and failing heart. Their expression is decreased in heart failure due to chronic overactivation of the sympathetic nervous system, contributing to declines in cardiac function and disease progression. Furthermore, therapies that prevent ß-adrenergic receptor downregulation or restore ß-adrenergic receptor levels are beneficial, making the determination of cardiac ß-adrenergic receptor expression in the heart an important consideration. Although quantitative RT-PCR can provide an indication of ß-adrenergic receptor density and subtype expression, mRNA levels do not always correlate with functional protein levels. Additionally, antibodies to ß-adrenergic receptors lack specificity, making immunoblotting and other antibody-based techniques unreliable. Radioligand binding assays were developed over 50 years ago and remain the gold standard for quantifying ß-adrenergic receptor densities in biological samples. This technique capitalizes on the binding of high-affinity, highly specific ligands to receptors and can give quantifiable levels of receptor expression. Furthermore, competition assays using subtype-selective antagonists generate binding profiles and can differentiate ß-adrenergic receptor subtype expression in cardiac tissue. This article focuses on the quantification of ß-adrenergic receptors in the heart using saturation and competition radioligand binding techniques to quantify ß-adrenergic receptor density and ligand affinities in cardiac membranes. © 2023 Wiley Periodicals LLC. Basic Protocol: Radioligand binding to quantify adrenergic receptor expression in the heart.

Vasovagal Syncope Is Associated with Variants in Genes Involved in Neurohumoral Signaling Pathways.

Vasovagal syncope (VVS) is the most common cause of sudden loss of consciousness. VVS results from cerebral hypoperfusion, due to abnormal autonomic control of blood circulation, leading to arterial hypotension. It is a complex disease, and its development is largely associated with genetic susceptibility. Since abnormal neurohumoral regulation plays an important role in VVS development, we analyzed the association of VVS with polymorphic variants of ADRA1A, ADRB1, HTR1A, ADORA2A, COMT, and NOS3 genes, the products of which are involved in neurohumoral signaling, in patients with a confirmed VVS diagnosis (157 subjects) and individuals without a history of syncope (161 subjects). We were able to identify the associations between VVS and alleles/genotypes ADRA1A rs1048101, ADRB1 rs1801253, ADORA2A rs5751876, and COMT rs4680, as well as NOS3 rs2070744 in biallelic combination with COMT rs4680. Thus, we are the first to observe, within a single study, the role of the genes that encode α- and ß-adrenergic receptors, catechol-O-methyltransferase, adenosine receptors and nitric oxide synthase in VVS development. These findings demonstrate that the genes involved in neurohumoral signaling pathways contribute to the formation of a genetic susceptibility to VVS.

Pharmacological activation of the C5a receptor leads to stimulation of the ß-adrenergic receptor and alleviates cognitive impairment in a murine model of familial Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease of the brain causing either familial or sporadic dementia. We have previously administered the modified C5a receptor agonist (EP67) for a short period to a transgenic mouse model of AD (5XFAD) and have observed not only reduction in ß-amyloid deposition and gliosis but also improvement in cognitive impairment. Inquiring, however, on the effects of EP67 in an already heavily burdened animal, thus representing a more realistic scenario, we treated 6-month-old 5XFAD mice for a period of 14 weeks. We recorded a significant decrease in both fibrillar and pre-fibrillar ß-amyloid as well as remarkable amelioration of cognitive impairment. Following proteomic analysis and pathway association, we postulate that these events are triggered through the upregulation of ß-adrenergic and GABAergic signaling. In summary, our results reveal how inflammatory responses can be employed in inducing tangible phenotype improvements even in advanced stages of AD.

Loss of adipose TET proteins enhances ß-adrenergic responses and protects against obesity by epigenetic regulation of ß3-AR expression.

ß-adrenergic receptor (ß-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished ß3-adrenergic receptor (ß3-AR) expression and decreased ß-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of ß3-AR expression in adipocytes, thereby attenuating the responsiveness to ß-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased ß3-AR expression and thereby enhanced the downstream ß-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning in vitro and in vivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of ß3-AR in both white and brown adipose tissues and remained sensitive to ß-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed ß3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-ß3-AR axis could be targeted to treat obesity and related metabolic diseases.

The Role of ß-Adrenergic Receptors in the Regulation of the Functions of Innate Immune Cells during Cold Stress In Vivo.

It was found that 10-min cold stress enhanced stimulated production of ROS, while 60-min cold stress increased both spontaneous and stimulated ROS production by peritoneal macrophages. ß-Adrenergic receptor blockade leveled the effect of 10-min stress in stimulated cultures and the effect of 60-min stress in spontaneous cultures. None variants of cold stress affected spontaneous and stimulated production of IL-1ß. We observed an increase in the production of IL-1ß in stimulated cultures from animals subjected to 10- and 60-min stress against the background of propranolol. At the same time, both variants of cold exposure, irrespective of ß-adrenergic receptor blockade, stimulated IL-10 synthesis in spontaneous and activated samples. None of the used models of cold exposure affected the phagocytic activity of peritoneal macrophages. Thus, ß-adrenergic receptors are directly involved in the regulation of cytokine production and microbicidal potential of macrophages in acute cold stress.

Single-nucleotide polymorphism of ADRß2 and CDKN1B genes in Egyptian patients with coronary artery in-stent restenosis.

BACKGROUND: In-stent restenosis is a common complication after percutaneous coronary intervention. The purpose of the current study is to look for associations of genetic variation in adrenergic beta-2 receptor (ADRß2), and cyclin-dependent kinase inhibitor 1B (CDKN1B) genes in patients diagnosed with in-stent restenosis (ISR) after percutaneous coronary intervention in the Egyptians. METHODS: Polymorphisms in ADRß2 and CDKN1B were determined using PCR-restriction fragment length polymorphism in 200 Egyptian patients who underwent coronary angioplasty and stent placement of whom 100 patients developed ISR. RESULTS: We found that the GG genotype of ADRß2 and CC genotype of CDKN1B were more likely to develop restenosis after stenting (odds ratio = 3.7 and 3.2; P = 0.001, respectively). Our study considered that male sex, diabetes, obesity, bare-metal stents type of implanted stents, longer stents, GG genotype of ADRß2, and CC genotype of CDK1B were significant independent predictors for ISR. CONCLUSION: our results indicate that ADRß2 (rs1042713) and CDKN1B (rs36228499) could be associated with the development of ISR in Egyptians.

Maoto, a traditional Japanese medicine, controls acute systemic inflammation induced by polyI:C administration through noradrenergic function.

Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a ß-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via ß-adrenergic receptors in vivo.

Dominant Role of PI3K p110α over p110ß in Insulin and ß-Adrenergic Receptor Signalling.

Attribution of specific roles to the two ubiquitously expressed PI 3-kinase (PI3K) isoforms p110α and p110ß in biological functions they have been implicated, such as in insulin signalling, has been challenging. While p110α has been demonstrated to be the principal isoform activated downstream of the insulin receptor, several studies have provided evidence for a role of p110ß. Here we have used isoform-selective inhibitors to estimate the relative contribution of each of these isoforms in insulin signalling in adipocytes, which are a cell type with essential roles in regulation of metabolism at the systemic level. Consistent with previous genetic and pharmacological studies, we found that p110α is the principal isoform activated downstream of the insulin receptor under physiological conditions. p110α interaction with Ras enhanced the strength of p110α activation by insulin. However, this interaction did not account for the selectivity for p110α over p110ß in insulin signalling. We also demonstrate that p110α is the principal isoform activated downstream of the ß-adrenergic receptor (ß-AR), another important signalling pathway in metabolic regulation, through a mechanism involving activation of the cAMP effector molecule EPAC1. This study offers further insights in the role of PI3K isoforms in the regulation of energy metabolism with implications for the therapeutic application of selective inhibitors of these isoforms.

Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute ß-Adrenergic Receptor Stimulation In Vivo.

ß-adrenergic receptor (ß-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic ß-AR agonist that targets both ß1-AR and ß2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to ß-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.

Investigating changes in ß-adrenergic gene expression (ADRB1 and ADRB2) in Takotsubo (stress) cardiomyopathy syndrome; a pilot study.

BACKGROUND: Takotsubo Cardiomyopathy (TC) is a rare disorder that is mostly caused by stress and is often misdiagnosed. We aimed to analyze Takotsubo Syndrome at the molecular level by using the Oxford Nanopore Minion Device and its protocol. METHODS AND RESULTS: Ten patients who were previously diagnosed with Takotsubo Syndrome (increased after decrease in ejection fraction and without critical stenosis in coronary arteries) and 10 healthy individuals in the control group were included in our project. The mean age was 53 ± 12.2 for the patient group and 52.4 ± 9.9 for the control group, and the left ventricular ejection fraction was 50.3 ± 11.5 for the patient group and 64.2 ± 2.8 for the control group (p < 0.05). Peripheral blood of patients and healthy individuals was taken and their DNA was obtained. By making long reads throughout the genome, the most studied regions responsible for ß-adrenergic signaling pathways; The gene expression level of cardiac ß-1 ADRB1 (rs1801253-ENST00000369295.4), G > C, (Gly389Arg) and cardiac ß-2 ADRB2 (rs1800888-ENSG00000169252), C > T, (Thr165Ile) adrenoceptors was investigated. As a result; no structural variation was detected leading to Takotsubo Cardiomyopathy. The results obtained from the bioinformatics analysis were also checked from the VarSome Tools and similar results were found. CONCLUSIONS: Many publications in TC susceptibility have that may lead to adrenergic pathway dysregulation, most studied adrenergic receptor genes in the similar literatures too. We searched for genetic variants in b1AR and b2AR genes in our study and however we could not find any variants in this study, we think larger numbers of cohort studies are needed.

The Association of Trp64Arg Polymorphism in the Beta-Adrenergic Receptor With Insulin Resistance: Meta-Analysis.

Background: Insulin resistance is a metabolic disorder that occurs in type 2 diabetes mellitus and obesity. Genetic factors such as ß3-adrenoceptor polymorphism (Trp64Arg) may be involved in IR and insulin secretion. However, their association is controversial. Therefore, the current meta-analysis was conducted to clarify the relationship between the Trp64Arg and IR. Methods: The literature search was performed in PubMed, Embase, and Web of Science using the keywords "Receptors, Adrenergic, beta-3, Receptors, Adrenergic, Insulin Resistance, Protein-Coupled Receptor Kinase 3" from 2005 to February 7, 2021. We used a random-effects model to calculate the pooled effect size. We conducted subgroup analysis and regression analysis to identify sources of heterogeneity; and Egger's test and funnel plot were used to test publication bias. Finally, we conducted a sensitivity analysis. Results: We included eight papers with 1,586 subjects. There was a positive correlation between Trp64Arg mutation and insulin level (standardized mean difference = 0.20, 95% confidence intervals: 0.00 to 0.39, I2 = 57.6%, p = 0.016). However, there was no association between Trp64Arg and the homeostasis model (HOMA-IR) assessment. Egger's tests showed no publication bias; the sensitivity analysis showed that our results were stable. Regression analysis revealed no source of heterogeneity. Conclusion: Trp64Arg may be associated with IR. European ancestry, obesity, plasma insulin level, and test status may be potential factors affecting the relationship between Trp64Arg and IR.

N-butylidenephthalide ameliorates high-fat diet-induced obesity in mice and promotes browning through adrenergic response/AMPK activation in mouse beige adipocytes.

Thermogenesis (non-exercise activity) in brown adipose tissue (BAT) promotes energy expenditure because of its higher number of mitochondria than white adipose tissue (WAT). The main function of thermogenesis in BAT can counteract obesity through the dissipation of calories as heat. N-butylidenephthalide (BP) is a natural derivative from Angelica sinensis, a Chinese herb that has been used for thousands of years. In this report, we demonstrated that BP improved the metabolic profiles of mice with high fat diet-induced obesity (DIO) by preventing weight gain, improving serum blood parameters, enhancing energy expenditure, stimulating white fat browning, and reversing hepatic steatosis. Further investigations demonstrated that BP administration upregulated the mRNA expression of beige (CD137, TMEM26) and brown fat selected genes (UCP1, PRDM16, PGC-1α, PPARγ) in white adipose tissues. In vitro studies, BP treatment increased multilocular lipid droplet levels, induced ß-adrenergic receptor (cAMP/PKA) and AMP-activated protein kinase (AMPK) signaling (AMPK/acetyl-CoA carboxylase/SIRT1), and increased oxygen consumption in murine differentiated beige adipocytes, and the effects of BP were blocked by an AMPK inhibitor. BP promoted the interaction of AMPK with PGC-1α in beige adipocytes. Our findings provide novel insights into the application of BP in regulating energy metabolism and suggest its utility for clinical use in the treatment of obesity and related diseases.

Characterization of ß-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with ß-adrenergic receptor agonists and antagonists.

Chinese hamster ovary cell constructs expressing either the ß 1-, ß 2- or ß 3-adrenergic receptor (AR) were used to determine whether a novel ß-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific ß-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the ß 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the ß 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the ß-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for ß 1-, ß2-, and ß 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; ß-AR pan-agonist), dobutamine hydrochloride (DOB; specific ß 1-AA), salbutamol sulfate (SAL; specific ß 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific ß 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for ß 1-, ß 2-, and ß 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant ß-AR mRNA in both adipose tissues was the ß 2-AR (P < 0.05), with the ß 1- and ß 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these ß-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the ß 1- and ß 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to ß-adrenergic ligands, especially those that are agonists at the ß 1- and ß3-receptor subtypes. The minimal mRNA expression of the ß 1- and ß 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these ß-AR subtypes.

The cAMP-phosphodiesterase 4 (PDE4) controls ß-adrenoceptor- and CFTR-dependent saliva secretion in mice.

Saliva, while often taken for granted, is indispensable for oral health and overall well-being, as inferred from the significant impairments suffered by patients with salivary gland dysfunction. Here, we show that treatment with several structurally distinct PAN-PDE4 inhibitors, but not a PDE3 inhibitor, induces saliva secretion in mice, indicating it is a class-effect of PDE4 inhibitors. In anesthetized mice, while neuronal regulations are suppressed, PDE4 inhibition potentiates a ß-adrenoceptor-induced salivation, that is ablated by the ß-blocker Propranolol and is absent from homozygous ΔF508-CFTR mice lacking functional CFTR. These data suggest that PDE4 acts within salivary glands to gate saliva secretion that is contingent upon the cAMP/PKA-dependent activation of CFTR. Indeed, PDE4 contributes the majority of total cAMP-hydrolytic capacity in submandibular-, sublingual-, and parotid glands, the three major salivary glands of the mouse. In awake mice, PDE4 inhibitor-induced salivation is reduced by CFTR deficiency or ß-blockers, but also by the muscarinic blocker Atropine, suggesting an additional, central/neuronal mechanism of PDE4 inhibitor action. The PDE4 family comprises four subtypes, PDE4A-D. Ablation of PDE4D, but not PDE4A-C, produced a minor effect on saliva secretion, implying that while PDE4D may play a predominant role, PDE4 inhibitor-induced salivation results from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data reveal a critical role for PDE4/PDE4D in controlling CFTR function in an in vivo model and in inducing salivation, hinting at a therapeutic potential of PDE4 inhibition for cystic fibrosis and conditions associated with xerostomia.

Reconstitution of ß-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of ß-adrenergic receptors (ß-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full ß-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of ß1-AR or ß2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of ß-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of ß1-AR versus ß2-AR activity. This system allows for the addressing of central unresolved issues in the ß-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Activation of the ß-adrenergic receptor exacerbates lipopolysaccharide-induced wasting of skeletal muscle cells by increasing interleukin-6 production.

The skeletal muscle mass has been shown to be affected by catecholamines, such as epinephrine (Epi), norepinephrine (NE), and isoproterenol (ISO). On the other hand, lipopolysaccharide (LPS), one of the causative substances of sepsis, induces muscle wasting via toll-like receptors expressed in skeletal muscle. Although catecholamines are frequently administered to critically ill patients, it is still incompletely understood how these drugs affect skeletal muscle during critical illness, including sepsis. Herein, we examined the direct effects of catecholamines on LPS-induced skeletal muscle wasting using the C2C12 myoblast cell line. Muscle wasting induced by catecholamines and/or LPS was analyzed by the use of the differentiated C2C12 myotubes, and its underlying mechanism was explored by immunoblotting analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and the TransAM kit for p-65 NF-κB. Epi augmented myosin heavy chain (MHC) protein loss and reduction of the myotube diameter induced by LPS. LPS induced C/EBPδ protein, Atrogin-1 and inteleukin-6 (IL-6), and these responses were potentiated by Epi. An IL-6 inhibitor, LMT28, suppressed the potentiating effect of Epi on the LPS-induced responses. NF-κB activity was induced by LPS, but was not affected by Epi and recombinant IL-6, and the NF-κB inhibitor, Bay 11-7082, abolished Atrogin-1 mRNA expression induced by LPS with or without Epi. NE and ISO also potentiated LPS-induced IL-6 and Atroign-1 mRNA expression. Carvedilol, a nonselective ß-adrenergic receptor antagonist, suppressed the facilitating effects of Epi on the Atrogin-1 mRNA induction by LPS, and abolished the effects of Epi on the MHC protein loss in the presence of LPS. It was concluded that Epi activates the ß-adrenergic receptors in C2C12 myotubes and the IL-6-STAT3 pathway, leading to the augmentation of LPS-induced activation of the NF-κB- C/EBPδ-Atrogin-1 pathway and to the exacerbation of myotube wasting.

Role of mTORC2 in biphasic regulation of brown fat metabolism in response to mild and severe cold.

Nonshivering thermogenesis is essential for mammals to maintain body temperature. According to the canonical view, temperature is sensed by cutaneous thermoreceptors and nerve impulses transmitted to the hypothalamus, which generates sympathetic signals to ß-adrenergic receptors in brown adipocytes. The energy for heat generation is primarily provided by the oxidation of fatty acids derived from triglyceride hydrolysis and cellular uptake. Fatty acids also activate the uncoupling protein, UCP1, which creates a proton leak that uncouples mitochondrial oxidative phosphorylation from ATP production, resulting in energy dissipation as heat. Recent evidence supports the idea that in response to mild cold, ß-adrenergic signals stimulate not only lipolysis and fatty acid oxidation, but also act through the mTORC2-Akt signaling module to stimulate de novo lipogenesis. This opposing anabolic effect is thought to maintain lipid fuel stores during increased catabolism. We show here, using brown fat-specific Gs-alpha knockout mice and cultured adipocytes that, unlike mild cold, severe cold directly cools brown fat and bypasses ß-adrenergic signaling to inhibit mTORC2. This cell-autonomous effect both inhibits lipogenesis and augments UCP1 expression to enhance thermogenesis. These findings suggest a novel mechanism for overriding ß-adrenergic-stimulated anabolic activities while augmenting catabolic activities to resolve the homeostatic crisis presented by severe cold.