Increased ß2-adrenergic signaling promotes fracture healing through callus neovascularization in mice.

Traumatic brain injury (TBI) leads to skeletal changes, including bone loss in the unfractured skeleton, and paradoxically accelerates healing of bone fractures; however, the mechanisms remain unclear. TBI is associated with a hyperadrenergic state characterized by increased norepinephrine release. Here, we identified the ß2-adrenergic receptor (ADRB2) as a mediator of skeletal changes in response to increased norepinephrine. In a murine model of femoral osteotomy combined with cortical impact brain injury, TBI was associated with ADRB2-dependent enhanced fracture healing compared with osteotomy alone. In the unfractured 12-week-old mouse skeleton, ADRB2 was required for TBI-induced decrease in bone formation and increased bone resorption. Adult 30-week-old mice had higher bone concentrations of norepinephrine, and ADRB2 expression was associated with decreased bone volume in the unfractured skeleton and better fracture healing in the injured skeleton. Norepinephrine stimulated expression of vascular endothelial growth factor A and calcitonin gene-related peptide-α (αCGRP) in periosteal cells through ADRB2, promoting formation of osteogenic type-H vessels in the fracture callus. Both ADRB2 and αCGRP were required for the beneficial effect of TBI on bone repair. Adult mice deficient in ADRB2 without TBI developed fracture nonunion despite high bone formation in uninjured bone. Blocking ADRB2 with propranolol impaired fracture healing in mice, whereas the ADRB2 agonist formoterol promoted fracture healing by regulating callus neovascularization. A retrospective cohort analysis of 72 patients with long bone fractures indicated improved callus formation in 36 patients treated with intravenous norepinephrine. These findings suggest that ADRB2 is a potential therapeutic target for promoting bone healing.

Impairment of the adrenergic reserve associated with exercise intolerance in a murine model of heart failure with preserved ejection fraction.

AIM: Exercise intolerance is the central symptom in patients with heart failure with preserved ejection fraction. In the present study, we investigated the adrenergic reserve both in vivo and in cardiomyocytes of a murine cardiometabolic HFpEF model. METHODS: 12-week-old male C57BL/6J mice were fed regular chow (control) or a high-fat diet and L-NAME (HFpEF) for 15 weeks. At 27 weeks, we performed (stress) echocardiography and exercise testing and measured the adrenergic reserve and its modulation by nitric oxide and reactive oxygen species in left ventricular cardiomyocytes. RESULTS: HFpEF mice (preserved left ventricular ejection fraction, increased E/e', pulmonary congestion [wet lung weight/TL]) exhibited reduced exercise capacity and a reduction of stroke volume and cardiac output with adrenergic stress. In ventricular cardiomyocytes isolated from HFpEF mice, sarcomere shortening had a higher amplitude and faster relaxation compared to control animals. Increased shortening was caused by a shift of myofilament calcium sensitivity. With addition of isoproterenol, there were no differences in sarcomere function between HFpEF and control mice. This resulted in a reduced inotropic and lusitropic reserve in HFpEF cardiomyocytes. Preincubation with inhibitors of nitric oxide synthases or glutathione partially restored the adrenergic reserve in cardiomyocytes in HFpEF. CONCLUSION: In this murine HFpEF model, the cardiac output reserve on adrenergic stimulation is impaired. In ventricular cardiomyocytes, we found a congruent loss of the adrenergic inotropic and lusitropic reserve. This was caused by increased contractility and faster relaxation at rest, partially mediated by nitro-oxidative signaling.

Unveiling the antinociceptive mechanisms of Methyl-2-(4-chloro-phenyl)-5-benzoxazoleacetate: insights from nociceptive assays in mice.

OBJECTIVE: Methyl-2-(4-chloro- phenyl)-5-benzoxazoleacetate (MCBA), a synthetic benzoxazole derivative with established antipsoriatic efficacy, was investigated for potential antinociceptive effects. This study employs various nociceptive assays in mice to elucidate MCBA's antinociceptive mechanisms. MATERIALS AND METHODS: MCBA's antinociceptive potential was tested against various nociception models induced by formalin, glutamate, capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor agonist, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator. It was then assessed using the hot plate test and examined within the acetic acid-induced writhing test. During the acetic acid-induced writhing test, MCBA was pre-challenged against selective receptor antagonists such as naloxone, caffeine, atropine, yohimbine, ondansetron, and haloperidol. It was also pre-challenged with ATP-sensitive potassium channel inhibitor (glibenclamide) to further elucidate its antinociceptive mechanism. RESULTS: The results showed that oral administration of MCBA led to a dose-dependent and significant inhibition (p < 0.05) of nociceptive effects across all evaluated models at doses of 60, 120, and 240 mg/kg. Moreover, the efficacy of MCBA's antinociceptive potential was significantly counteracted (p < 0.0001) by specific antagonists: (i) directed at adenosinergic, alpha-2 adrenergic, and cholinergic receptors using caffeine, yohimbine, and atropine, respectively; and (ii) targeting ATP-sensitive potassium channels, employing glibenclamide. Antagonists aimed at opioidergic and serotoninergic receptors (naloxone and ondansetron, respectively) had poor utility in inhibiting antinociceptive activity. Conversely, the dopaminergic receptor antagonist haloperidol potentiated locomotor abnormalities associated with MCBA treatment. CONCLUSIONS: MCBA-induced antinociception involves modulation of glutamatergic-, TRVP1 receptors- and PKC-signaling pathways. It impacts adenosinergic, alpha-2 adrenergic, and cholinergic receptors and opens ATP-sensitive potassium channels.

Specific pharmacological and Gi/o protein responses of some native GPCRs in neurons.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and are important drug targets. The discovery of drugs targeting these receptors and their G protein signaling properties are based on assays mainly performed with modified receptors expressed in heterologous cells. However, GPCR responses may differ in their native environment. Here, by using highly sensitive Gi/o sensors, we reveal specific properties of Gi/o protein-mediated responses triggered by GABAB, α2 adrenergic and cannabinoid CB1 receptors in primary neurons, different from those in heterologous cells. These include different profiles in the Gi/o protein subtypes-mediated responses, and differences in the potencies of some ligands even at similar receptor expression levels. Altogether, our results show the importance of using biosensors compatible with primary cells for evaluating the activities of endogenous GPCRs in their native environment.

Peri-operative individually tailored psychological intervention in breast cancer patients improves psychological indices and molecular biomarkers of metastasis in excised tumors.

Perioperative stress and inflammatory signaling can invigorate pro-metastatic molecular processes in patients' tumors, potentially worsening long-term survival. Yet, it is unknown whether pre-operative psychotherapeutic interventions can attenuate such effects. Herein, three weeks before surgery, forty women diagnosed with stage I-III invasive ductal/lobular breast carcinoma were randomized to a 6-week one-on-one psychological intervention (6 meetings with a medical psychologist and bi-weekly phone calls) versus standard nursing-staff-attention. The intervention protocol was individually tailored based on evaluation of patients' emotional, cognitive, physiological, and behavioral stress response-patterns, and also included psychoeducation regarding medical treatments and recruitment of social support. Resected primary tumors were subjected to whole-genome RNA sequencing and bioinformatic analyses, assessing a priori hypothesized cancer-relevant molecular signatures. Self-report questionnaires (BSI-18, Hope-18, MSPSS, and a stress-scale) were collected three (T1) and one (T2) week before surgery, a day before (T3) and after (T4) surgery, and three weeks (T5) and 3-months (T6) following surgery. The intervention reduced distress (GSI), depression, and somatization scores (BSI-18: p < 0.01, p < 0.05, p < 0.05; T5 vs. T1). Additionally, tumors from treated patients (vs. controls) showed: (i) decreased activity of transcription control pathways involved in adrenergic and glucocorticoid signaling (CREB, GR) (p < 0.001), pro-inflammatory signaling (NFkB) (p < 0.01), and pro-malignant signaling (ETS1, STAT and GATA families) (p < 0.001, p < 0.01, p < 0.005); (ii) increased M1 macrophage polarization (p < 0.05), and CD4+ T cell activity (p < 0.01); and an unexpected increase in epithelial-to-mesenchymal-transition (EMT) signature (p < 0.005). This is the first randomized controlled trial to show beneficial effects of a psychological perioperative intervention on tumor pro-metastatic molecular biomarkers.

Prolonged FGF21 treatment increases energy expenditure and induces weight loss in obese mice independently of UCP1 and adrenergic signaling.

Fibroblast growth factor 21 (FGF21) reduces body weight, which was attributed to induced energy expenditure (EE). Conflicting data have been published on the role of uncoupling protein 1 (UCP1) in this effect. Therefore, we aimed to revisit the thermoregulatory effects of FGF21 and their implications for body weight regulation. We found that an 8-day treatment with FGF21 lowers body weight to similar extent in both wildtype (WT) and UCP1-deficient (KO) mice fed high-fat diet. In WT mice, this effect is solely due to increased EE, associated with a strong activation of UCP1 and with excess heat dissipated through the tail. This thermogenesis takes place in the interscapular region and can be attenuated by a ß-adrenergic inhibitor propranolol. In KO mice, FGF21-induced weight loss correlates with a modest increase in EE, which is independent of adrenergic signaling, and with a reduced energy intake. Interestingly, the gene expression profile of interscapular brown adipose tissue (but not subcutaneous white adipose tissue) of KO mice is massively affected by FGF21, as shown by increased expression of genes encoding triacylglycerol/free fatty acid cycle enzymes. Thus, FGF21 elicits central thermogenic and pyretic effects followed by a concomitant increase in EE and body temperature, respectively. The associated weight loss is strongly dependent on UCP1-based thermogenesis. However, in the absence of UCP1, alternative mechanisms of energy dissipation may contribute, possibly based on futile triacylglycerol/free fatty acid cycling in brown adipose tissue and reduced food intake.

Integrated miRNA-mRNA networks underlie attenuation of chronic ß-adrenergic stimulation-induced cardiac remodeling by minocycline.

Adverse cardiac remodeling contributes to heart failure development and progression, partly due to inappropriate sympathetic nervous system activation. Although ß-adrenergic receptor (ß-AR) blockade is a common heart failure therapy, not all patients respond, prompting exploration of alternative treatments. Minocycline, an FDA-approved antibiotic, has pleiotropic properties beyond antimicrobial action. Recent evidence suggests it may alter gene expression via changes in miRNA expression. Thus, we hypothesized that minocycline could prevent adverse cardiac remodeling induced by the ß-AR agonist isoproterenol, involving miRNA-mRNA transcriptome alterations. Male C57BL/6J mice received isoproterenol (30 mg/kg/day sc) or vehicle via osmotic minipump for 21 days, along with daily minocycline (50 mg/kg ip) or sterile saline. Isoproterenol induced cardiac hypertrophy without altering cardiac function, which minocycline prevented. Total mRNA sequencing revealed isoproterenol altering gene networks associated with inflammation and metabolism, with fibrosis activation predicted by integrated miRNA-mRNA sequencing, involving miR-21, miR-30a, miR-34a, miR-92a, and miR-150, among others. Conversely, the cardiac miRNA-mRNA transcriptome predicted fibrosis inhibition in minocycline-treated mice, involving antifibrotic shifts in Atf3 and Itgb6 gene expression associated with miR-194 upregulation. Picrosirius red staining confirmed isoproterenol-induced cardiac fibrosis, prevented by minocycline. These results demonstrate minocycline's therapeutic potential in attenuating adverse cardiac remodeling through miRNA-mRNA-dependent mechanisms, especially in reducing cardiac fibrosis. NEW & NOTEWORTHY We demonstrate that minocycline treatment prevents cardiac hypertrophy and fibrotic remodeling induced by chronic ß-adrenergic stimulation by inducing antifibrotic shifts in the cardiac miRNA-mRNA transcriptome.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

Cerebral sympatholysis: experiments on in vivo cerebrovascular regulation and ex vivo cerebral vasomotor control.

Whether cerebral sympathetic-mediated vasomotor control can be modulated by local brain activity remains unknown. This study tested the hypothesis that the application or removal of a cognitive task during a cold pressor test (CPT) would attenuate and restore decreases in cerebrovascular conductance (CVC), respectively. Middle cerebral artery blood velocity (transcranial Doppler) and mean arterial pressure (finger photoplethysmography) were examined in healthy adults (n = 16; 8 females and 8 males) who completed a control CPT, followed by a CPT coupled with a cognitive task administered either 1) 30 s after the onset of the CPT and for the duration of the CPT or 2) at the onset of the CPT and terminated 30 s before the end of the CPT (condition order was counterbalanced). The major finding was that the CPT decreased the index of CVC, and such decreases were abolished when a cognitive task was completed concurrently and restored when the cognitive task was removed. As a secondary experiment, vasomotor interactions between sympathetic transduction pathways (α1-adrenergic and Y1-peptidergic) and compounds implicated in cerebral blood flow control [adenosine, and adenosine triphosphate (ATP)] were explored in isolated porcine cerebral arteries (wire myography). The data reveal α1-receptor agonism potentiated vasorelaxation modestly in response to adenosine, and preexposure to ATP attenuated contractile responses to α1-agonism. Overall, the data suggest a cognitive task attenuates decreases in CVC during sympathoexcitation, possibly related to an interaction between purinergic and α1-adrenergic signaling pathways.NEW & NOTEWORTHY The present study demonstrates that the cerebrovascular conductance index decreases during sympathoexcitation and this response can be positively and negatively modulated by the application or withdrawal of a nonexercise cognitive task. Furthermore, isolated vessel experiments reveal that cerebral α1-adrenergic agonism potentiates adenosine-mediated vasorelaxation and ATP attenuates α1-adrenergic-mediated vasocontraction.

Functional and 123I-MIBG scintigraphy assessment of cardiac adrenergic dysfunction in diabetes.

OBJECTIVES: To assess the agreement between clinical cardiovascular adrenergic function and cardiac adrenergic innervation in type 2 diabetes patients (T2D). METHODS: Thirty-three patients with T2D were investigated bimodally through (1) a standardized clinical cardiovascular adrenergic assessment, evaluating adequacy of blood pressure responses to the Valsalva maneuver and (2) 123I-meta-iodobenzylguanidine (MIBG) scintigraphy assessing myocardial adrenergic innervation measured as early and delayed heart heart/mediastinum (H/M) ratio, and washout rate (WR). RESULTS: T2D patients had significantly lower early and delayed H/M-ratios, and lower WR, compared to laboratory specific reference values. Thirteen patients had an abnormal adrenergic composite autonomic severity score (CASS > 0). Patients with abnormal CASS scores had significantly higher early H/M ratios (1.76 [1.66-1.88] vs. 1.57 [1.49-1.63], p < 0.001), higher delayed H/M ratios (1.64 [1.51:1.73] vs. 1.51 [1.40:1.61] (p = 0.02)), and lower WR (-0.13(0.10) vs -0.05(0.07), p = 0.01). Lower Total Recovery and shorter Pressure Recovery Time responses from the Valsalva maneuver was significantly correlated to lower H/M early (r = 0.55, p = 0.001 and r = 0.5, p = 0.003, respectively) and lower WR for Total Recovery (r = -0.44, p = 0.01). CONCLUSION: The present study found impairment of sympathetic innervation in T2D patients based on parameters derived from MIBG cardiac scintigraphy (low early H/M, delayed H/M, and WR). These results confirm prior studies. We found a mechanistically inverted relationship with favourable adrenergic cardiovascular responses being significantly associated unfavourable MIBG indices for H/M early and delayed. This paradoxical relationship needs to be further explored but could indicate adrenergic hypersensitivity in cardiac sympathetic denervated T2D patients.

Myofilament-based physiological regulatory compensation preserves diastolic function in failing hearts with severe Ca2+ handling deficits.

Severe dysfunction in cardiac muscle intracellular Ca2+ handling is a common pathway underlying heart failure. Here we used an inducible genetic model of severe Ca2+ cycling dysfunction by the targeted temporal gene ablation of the cardiac Ca2+ ATPase, SERCA2, in otherwise normal adult mice. In this model, in vivo heart performance was minimally affected initially, even though Serca2a protein was markedly reduced. The mechanism underlying the sustained in vivo heart performance in the weeks prior to complete heart pump failure and death is not clear and is important to understand. Studies were primarily focused on understanding how in vivo diastolic function could be relatively normal under conditions of marked Serca2a deficiency. Interestingly, data show increased cardiac troponin I (cTnI) serine 23/24 phosphorylation content in hearts upon Serca2a ablation in vivo. We report that hearts isolated from the Serca2-deficient mice retained near normal heart pump functional responses to ß-adrenergic stimulation. Unexpectedly, using genetic complementation models, in concert with inducible Serca2 ablation, data show that Serca2a-deficient hearts that also lacked the central ß-adrenergic signaling-dependent Serca2a negative regulator, phospholamban (PLN), had severe diastolic dysfunction that could still be corrected by ß-adrenergic stimulation. Notably, integrating a serines 23/24-to-alanine PKA-refractory sarcomere incorporated cTnI molecular switch complex in mice deficient in Serca2 showed blunting of ß-adrenergic stimulation-mediated enhanced diastolic heart performance. Taken together, these data provide evidence of a compensatory regulatory role of the myofilaments as a critical physiological bridging mechanism to aid in preserving heart diastolic performance in failing hearts with severe Ca2+ handling deficits.

Neuromorphic electro-stimulation based on atomically thin semiconductor for damage-free inflammation inhibition.

Inflammation, caused by accumulation of inflammatory cytokines from immunocytes, is prevalent in a variety of diseases. Electro-stimulation emerges as a promising candidate for inflammatory inhibition. Although electroacupuncture is free from surgical injury, it faces the challenges of imprecise pathways/current spikes, and insufficiently defined mechanisms, while non-optimal pathway or spike would require high current amplitude, which makes electro-stimulation usually accompanied by damage and complications. Here, we propose a neuromorphic electro-stimulation based on atomically thin semiconductor floating-gate memory interdigital circuit. Direct stimulation is achieved by wrapping sympathetic chain with flexible electrodes and floating-gate memory are programmable to fire bionic spikes, thus minimizing nerve damage. A substantial decrease (73.5%) in inflammatory cytokine IL-6 occurred, which also enabled better efficacy than commercial stimulator at record-low currents with damage-free to sympathetic neurons. Additionally, using transgenic mice, the anti-inflammation effect is determined by ß2 adrenergic signaling from myeloid cell lineage (monocytes/macrophages and granulocytes).

ß2-Adrenoceptor Activation Favor Acquisition of Tumorigenic Properties in Non-Tumorigenic MCF-10A Breast Epithelial Cells.

Noradrenaline and adrenaline, and their cognate receptors, are currently accepted to participate in cancer progression. They may also participate in cancer initiation, although their role in this phase is much less explored. The aim of this work was to study the influence of adrenergic stimulation in several processes related to breast cancer carcinogenesis, using several adrenergic agonists in the MCF-10A non-tumorigenic breast cells. Activation of the ß-adrenoceptors promoted an epithelial phenotype in MCF-10A cells, revealed by an increased expression of the epithelial marker E-cadherin and a decrease in the mesenchymal markers, N-cadherin and vimentin. MCF-10A cell motility and migration were also impaired after the ß-adrenoceptors activation. Concomitant with this effect, ß-adrenoceptors decrease cell protrusions (lamellipodia and filopodia) while increasing cell adhesion. Activation of the ß-adrenoceptors also decreases MCF-10A cell proliferation. When the MCF-10A cells were cultured under low attachment conditions, activation the of ß- (likely ß2) or of α2-adrenoceptors had protective effects against cell death, suggesting a pro-survival role of these adrenoceptors. Overall, our results showed that, in breast cells, adrenoceptor activation (mainly through ß-adrenoceptors) may be a risk factor in breast cancer by inducing some cancer hallmarks, providing a mechanistic explanation for the increase in breast cancer incidences that may be associated with conditions that cause massive adrenergic stimulation, such as stress.

Serotonergic and Adrenergic Neuroreceptor Manipulation Ameliorates Core Symptoms of ADHD through Modulating Dopaminergic Receptors in Spontaneously Hypertensive Rats.

The core symptoms of attention deficit hyperactivity disorder (ADHD) are due to the hypofunction of the brain's adrenergic (NE) and dopamine (DA) systems. Drugs that enhance DA and NE neurotransmission in the brain by blocking their transporters or receptors are the current therapeutic strategies. Of late, the emerging results point out the serotonergic (5-HT) system, which indirectly modulates the DA activity in reducing the core symptoms of ADHD. On this basis, second-generation antipsychotics, which utilize 5-HT receptors, were prescribed to children with ADHD. However, it is not clear how serotonergic receptors modulate the DA activity to minimize the symptoms of ADHD. The present study investigates the efficacy of serotonergic and alpha-2 adrenergic receptor manipulation in tackling the core symptoms of ADHD and how it affects the DA neuroreceptors in the brain regions involved in ADHD. Fifteen-day-old male spontaneously hypertensive rats (SHRs) received 5-HT1A agonist (ipsapirone) or 5-HT2A antagonist (MDL 100907) (i.p.) or alpha-2 agonist (GFC) from postnatal days 15 to 42 along with age-matched Wistar Kyoto rats (WKY) (n = 8 in each group). ADHD-like behaviors were assessed using a battery of behavioral tests during postnatal days 44 to 65. After the behavioral tests, rat brains were processed to estimate the density of 5-HT1A, 5-HT2A, DA-D1, and DA-D2 neuroreceptors in the prefrontal cortex, the striatum, and the substantia nigra. All three neuroreceptor manipulations were able to minimize the core symptoms of ADHD in SHRs. The positive effect was mainly associated with the upregulation of 5-HT2A receptors in all three areas investigated, while 5-HT1A was in the prefrontal cortex and the substantia nigra. Further, the DA-D1 receptor expression was downregulated by all three neuroreceptor manipulations except for alpha-2 adrenergic receptor agonists in the striatum and 5-HT2A antagonists in the substantia nigra. The DA-D2 expression was upregulated in the striatum while downregulated in the prefrontal cortex and the substantia nigra. In this animal model study, the 5-HT1A agonist or 5-HT2A antagonist monotherapies were able to curtail the ADHD symptoms by differential expression of DA receptors in different regions of the brain.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial ß1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac ß-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to ß-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the ß-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to ß1-AR (but not ß2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the ß1-AR-mediated responses was prevented by selective ß3-AR antagonists as well as inhibitors of Gi protein, Gßγ, G protein-coupled receptor kinase 2/3, or ß-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to ß-AR activation. Thus, 25HC can suppress the ß1-AR-dependent effects via engaging ß3-AR, Gi protein, Gßγ, G protein-coupled receptor kinase, and ß-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial ß-adrenergic signaling.

Sympathetic nerve-enteroendocrine L cell communication modulates GLP-1 release, brain glucose utilization, and cognitive function.

Glucose homeostasis is controlled by brain-gut communications. Yet our understanding of the neuron-gut interface in the glucoregulatory system remains incomplete. Here, we find that sympathetic nerves elevate postprandial blood glucose but restrict brain glucose utilization by repressing the release of glucagon-like peptide-1 (GLP-1) from enteroendocrine L cells. Sympathetic nerves are in close apposition with the L cells. Importantly, sympathetic denervation or intestinal deletion of the adrenergic receptor α2 (Adra2a) augments postprandial GLP-1 secretion, leading to reduced blood glucose levels and increased brain glucose uptake. Conversely, sympathetic activation shows the opposite effects. At the cellular level, adrenergic signaling suppresses calcium flux to limit GLP-1 secretion upon sugar ingestion. Consequently, abrogation of adrenergic signal results in a significant improvement in learning and memory ability. Together, our results reveal a sympathetic nerve-enteroendocrine unit in constraining GLP-1 secretion, thus providing a therapeutic nexus of mobilizing endogenous GLP-1 for glucose management and cognitive improvement.

A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.

"Photo-Adrenalines": Photoswitchable ß2 -Adrenergic Receptor Agonists as Molecular Probes for the Study of Spatiotemporal Adrenergic Signaling.

ß2 -adrenergic receptor (ß2 -AR) agonists are used for the treatment of asthma and chronic obstructive pulmonary disease, but also play a role in other complex disorders including cancer, diabetes and heart diseases. As the cellular and molecular mechanisms in various cells and tissues of the ß2 -AR remain vastly elusive, we developed tools for this investigation with high temporal and spatial resolution. Several photoswitchable ß2 -AR agonists with nanomolar activity were synthesized. The most potent agonist for ß2 -AR with reasonable switching is a one-digit nanomolar active, trans-on arylazopyrazole-based adrenaline derivative and comprises valuable photopharmacological properties for further biological studies with high structural accordance to the native ligand adrenaline.

Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.