Catecholamines modulate the hypoxic ventilatory response of larval zebrafish (Danio rerio).

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent ß-adrenoreceptor (AR)-mediated increase in fV that does not require expression of ß1-ARs, as the hyperventilatory response to ß-AR stimulation was unaltered in adrb1-/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min-1 without propranolol to 68±17 breaths min-1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1-/- mutants. Thus, ß1-AR is not required for the HVR and other ß-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.

Selectivity and Maximum Response of Vibegron and Mirabegron for ß3-Adrenergic Receptors.

Background: The ß3-adrenergic agonists vibegron and mirabegron have shown favorable safety profiles and efficacy for the treatment of overactive bladder. However, ß-adrenergic receptors are also found outside the bladder, which could lead to off-target activity. Objective: This study assessed the selectivity of vibegron and mirabegron for ß-adrenergic receptors and the maximal effect and potency for ß3-adrenergic receptors. Methods: Functional cellular assays were performed using Chinese hamster ovary-K1 cells expressing ß1-, Chinese hamster ovary cells expressing ß2-, and human embryonic kidney 293 cells expressing ß3-adrenergic receptors. Cells were incubated with vibegron, mirabegron, or control (ß1 and ß3, isoproterenol; ß2, procaterol). Responses were quantified using homogeneous time-resolved fluorescence of cyclic adenosine monophosphate and were normalized to the respective control. Half-maximal effective concentration and maximum response values were determined by nonlinear least-squares regression analysis. Results: Activation of ß3-adrenergic receptors with vibegron or mirabegron resulted in concentration-dependent ß3-adrenergic receptor responses. Mean (SEM) half-maximal effective concentration values at ß3-adrenergic receptors were 2.13 (0.25) nM for vibegron and 10.0 (0.56) nM for mirabegron. At a concentration of 10 µM, ß3-adrenergic activity relative to isoproterenol was 104% for vibegron and 88% for mirabegron. Maximum response at ß3-adrenergic receptors was 99.2% for vibegron and 80.4% for mirabegron. ß1-adrenergic activity was 0% and 3% for vibegron and mirabegron, respectively; ß2-adrenergic activity was 2% and 15%, respectively. Conclusions: Vibegron showed no measurable ß1 and low ß2 activity compared with mirabegron, which showed low ß1 and some ß2 activity. Both showed considerable selectivity at ß3-adrenergic receptors; however, vibegron demonstrated near-exclusive ß3 activity and a higher maximum ß3 response.

Acute Exercise-Induced Circulating Haematopoietic Stem and Progenitor Cells in Cardiac Patients - A Case Series.

BACKGROUND: Exercise-induced circulating haematopoietic stem and progenitor cell (HPC) number has been discussed in the context of regeneration in heart disease patients. OBJECTIVE: The aim of this pilot study was to compare the effect of different exercise protocols usually applied in cardiac rehabilitation on the number of acute, exercise-induced HPCs, related to potential mediators, e.g. biomarkers of sympathetic and oxidative stress, and inflammation. METHODS: This is a case series comprising seven patients suffering from coronary heart disease (CHD) undertaken at the Center for Ambulant Cardiac Rehabilitation. Patients (n=6) performed two exercise modes (constant-load, CLE; high-intensity interval, HIIE) in randomised order. Venous blood was drawn before and immediately after each test to assess CD34+/CD45+ HPC number by flow cytometry and biomarkers in blood plasma. The primary outcome was the change in HPC number, the secondary outcomes were changes in sympathetic/oxidative stress and markers of inflammation. RESULTS: Both exercise modes resulted in a non-significant increase in HPC number after exercise, even when the results of both tests were combined. Overall, free norepinephrine increased significantly and was positively related to exercise-induced HPC number (r=0.70, p<0.05). Markers of sympathetic activation (fNE), oxidative stress (myeloperoxidase) and inflammation (interleukin-6) significantly increased after CLE and HIIE with no difference between tests. CONCLUSIONS: Interestingly, acute CLE and HIIE did not stimulate significant HPC mobilisation in CHD, although both exercise modes elevated circulating concentrations of sympathetic activation. Haematopoietic stem and progenitor cell mobilisation could be blunted due to disease-related bone-marrow exhaustion.

Epinephrine Stimulates Cell Proliferation and Induces Chemoresistance in Myeloma Cells through the ß-Adrenoreceptor in vitro.

OBJECTIVES: To explore the effect of the ß-adrenoreceptor signaling pathway on myeloma cells. METHODS: The myeloma U266 cell line was treated with epinephrine and propranolol. Cell proliferation was analyzed by MTS assay. Apoptosis was detected by flow cytometry. The ß-receptor subtype and the key enzyme of epinephrine were identified by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Epinephrine (5-50 µM) promoted U266 cell growth in a dose-dependent manner and neutralized the inhibition effect of bortezomib (25 and 50 ng/mL) in vitro. Cell proliferation was inhibited by a ß-receptor antagonist, propranolol, at a concentration of 50-200 µM. The proportions of early and late apoptotic cells were enhanced after treatment with propranolol. The expression of caspase 3/7, 8, and 9 was elevated in propranolol-treated myeloma cells. Both ß1- and ß2-adrenoceptor mRNAs were expressed in the U266 cell line. Key enzymes dopamine hydroxylase and tyrosinehydroxylase were identified in myeloma cells. CONCLUSIONS: Our results reveal that epinephrine stimulates myeloma cell growth in vitro while the ß-blocker propranolol has an antiproliferative effect, indicating that stress hormones may trigger the progression of myeloma.

Mechanisms of PVP-functionalized silver nanoparticle toxicity in fish: Intravascular exposure disrupts cardiac pacemaker function and inhibits Na+/K+-ATPase activity in heart, but not gill.

Polyvinylpyrrolidone-functionalized silver nanoparticles (nAgPVP) are popular in consumer products for their colloidal stability and antimicrobial activity. Whole lake additions of nAgPVP cause long term, ecosystem-scale changes in fish populations but the mechanisms underlying this effect are unclear. We have previously shown that in fish, nAgPVP impairs cardiac contractility and Na+/K+-ATPase (NKA) activity in vitro, raising the possibility that heart dysfunction could underlie population-level exposure effects. The goal of this study was to determine if nAgPVP influences the control of heart rate (fh), blood pressure, or cardiac NKA activity in vivo. First, a dose-response curve for the effects of 5 nm nAgPVP on contractility was completed on isometrically contracting ventricular muscle preparations from Arctic char (Salvelinus alpinus) and showed that force production was lowest at 500 µg L-1 and maximum pacing frequency increased with nAgPVP concentration. Stroke volume, cardiac output, and power output were maintained in isolated working heart preparations from brook char (Salvelinus fontinalis) exposed to 700 µg L-1 nAgPVP. Both fh and blood pressure were elevated after 24 h in brook char injected with 700 µg kg body mass-1 nAgPVP and fh was insensitive to modulation with blockers of ß-adrenergic and muscarinic cholinergic receptors. Na+/K+-ATPase activity was significantly lower in heart, but not gill of nAgPVP injected fish. The results indicate that nAgPVP influences cardiac function in vivo by disrupting regulation of the pacemaker and cardiomyocyte ionoregulation. Impaired fh regulation may prevent fish from appropriately responding to environmental or social stressors and affect their ability to survive.

Oral propranolol for retinopathy of prematurity: risks, safety concerns, and perspectives.

OBJECTIVE: To evaluate safety and efficacy of oral propranolol administration in preterm newborns affected by an early phase of retinopathy of prematurity (ROP). STUDY DESIGN: Fifty-two preterm newborns with Stage 2 ROP were randomized to receive oral propranolol (0.25 or 0.5 mg/kg/6 hours) added to standard treatment or standard treatment alone. To evaluate safety of the treatment, hemodynamic and respiratory variables were continuously monitored, and blood samples were collected weekly to check for renal, liver, and metabolic balance. To evaluate efficacy of the treatment, the progression of the disease (number of laser treatments, number of bevacizumab treatments, and incidence of retinal detachment) was evaluated by serial ophthalmologic examinations, and plasma soluble E-selectin levels were measured weekly. RESULTS: Newborns treated with propranolol showed less progression to Stage 3 (risk ratio 0.52; 95% CI 0.47-0.58, relative reduction of risk 48%) or Stage 3 plus (relative risk 0.42 95% CI 0.31-0.58, relative reduction of risk 58%). The infants required fewer laser treatments and less need for rescue treatment with intravitreal bevacizumab (relative risk 0.48; 95% CI 0.29-0.79, relative reduction of risk 52 %), a 100% relative reduction of risk for progression to Stage 4. They also had significantly lower plasma soluble E-selectin levels. However, 5 of the 26 newborns treated with propranolol had serious adverse effects (hypotension, bradycardia), in conjunction with episodes of sepsis, anesthesia induction, or tracheal stimulation. CONCLUSION: This pilot study suggests that the administration of oral propranolol is effective in counteracting the progression of ROP but that safety is a concern.

Blocking Ocular Sympathetic Activity Inhibits Choroidal Neovascularization.

Purpose: To investigate how modulating ocular sympathetic activity affects progression of choroidal neovascularization (CNV), a hallmark feature of wet age-related macular degeneration (AMD). Methods: In the first of two studies, Brown Norway rats underwent laser-induced CNV and were assigned to one of the following groups: daily eye drops of artificial tears (n = 10; control group); daily eye drops of the ß-adrenoreceptor agonist isoproterenol (n = 10); daily eye drops of the ß-adrenoreceptor antagonist propranolol (n = 10); sympathetic internal carotid nerve (ICN) transection 6 weeks prior to laser-induced CNV (n = 10). In the second study, rats underwent laser-induced CNV followed by ICN transection at different time points: immediately after the laser injury (n = 6), 7 days after the laser injury (n = 6), and sham surgery 7 days after the laser injury (n = 6; control group). All animals were euthanized 14 days after laser application. CNV development was quantified with fluorescein angiography and optical coherence tomography (in vivo), as well as lesion volume analysis using 3D confocal reconstruction (postmortem). Angiogenic growth factor protein levels in the choroid were measured with ELISA. Results: In the first study, blocking ocular sympathetic activity through pharmacological or surgical manipulation led to a 75% or 70% reduction in CNV lesion volume versus the control group, respectively (P < 0.001). Stimulating ocular sympathetic activity with isoproterenol also led to a reduction in lesion volume, but only by 27% versus controls (P < 0.05). VEGF protein levels in the choroid were elevated in the three treatment groups (P < 0.01). In the second study, fluorescein angiography and CNV lesion volume analysis indicated that surgically removing the ocular sympathetic supply inhibited progression of laser-induced CNV, regardless of whether ICN transection was performed on the same day or 7 days after the laser injury. Conclusion: Surgical and pharmacological block of ocular sympathetic activity can inhibit progression of CNV in a rat model. Therefore, electrical block of ICN activity could be a potential bioelectronic medicine strategy for treating wet AMD.

Ractopamine residue in meat might protect people from Parkinson disease.

There is still no curative treatment for the exasperating Parkinson disease, the second most common neurodegenerative disorder. Intracytoplasmic Lewy body composing of phosphorylated α-synuclein in dopaminergic neuronal cells has been recognized as the characteristic pathologic change and believed to be the cause of neuronal cell loss in Parkinson disease. Recently, ß-adrenoreceptor antagonist was found to be correlated with an increasing incidence of Parkinson disease and ß-adrenoreceptor agonist, capable of inhibiting gene expression of α-synuclein, was associated with a reduced incidence of it. Therefore, a hypothesis is raised that ractopamine, a ß-adrenoreceptor agonist used as feed additive for increasing leanness of finishing cattle and swine, might provide protective effects for Parkinson disease and lower its incidence in the population consuming meat containing ractopamine residue.

Lower Sympathetic Nervous System Density and ß-adrenoreceptor Expression Are Involved in Gastric Cancer Progression.

BACKGROUND/AIM: Identifying the role of the sympathetic nervous system (SNS) in tumor progression is among the most important challenges in cancer research. This study aimed to investigate the role of the SNS and ß-adrenoreceptor in gastric cancer progression. MATERIALS AND METHODS: The density of SNS was quantified by immunohistochemical staining for tyrosine hydroxylase in 115 surgically-resected gastric cancer specimens. Immunostaining for ß1- and ß2-adrenoreceptor was also performed to examine the ß-adrenoreceptor expression status in gastric cancer. Then the association of protein expression status with histological grade, pathological tumor stage (pT), and pathological node stage of gastric cancer was investigated. RESULTS: The SNS density of pT4 tumors was significantly lower than that of pT1-3 tumors. The SNS density was positively correlated with ß1-adrenoreceptor expression status. In addition, lower ß1-adrenoreceptor expression was significantly associated with increased lymph node metastasis. Reduced ß2-adrenoreceptor staining proportion was significantly associated with worse histological grade. Furthermore, the proportion of ß2-adrenoreceptor staining was significantly lower in tumors with diffuse-type histology, than those with intestinal-type histology. CONCLUSION: A lower SNS density and ß-adrenoreceptor expression was associated with an aggressive oncogenic behavior including worse histological grade, advanced pT, and increased lymph node metastasis. SNS and ß-adrenergic pathway are involved in the negative regulation of gastric cancer progression.

Pharmacological Stimulation of Mitochondrial Biogenesis Using the Food and Drug Administration-Approved ß2-Adrenoreceptor Agonist Formoterol for the Treatment of Spinal Cord Injury.

A hallmark of the progressive cascade of damage referred to as secondary spinal cord injury (SCI) is vascular disruption resulting in decreased oxygen delivery and loss of mitochondria homeostasis. While therapeutics targeting restoration of single facets of mitochondrial function have proven largely ineffective clinically post-SCI, comprehensively addressing mitochondrial function via pharmacological stimulation of mitochondrial biogenesis (MB) is an underexplored strategy. This study examined the effects of formoterol, a mitochondrial biogenic Food and Drug Administration-approved selective and potent ß2-adrenoreceptor (ADRB2) agonist, on recovery from SCI in mice. Female C57BL/6 mice underwent moderate SCI using a force-controlled impactor-induced contusion model, followed by daily formoterol intraperitoneal administration (0.1 mg/kg) beginning 1 h post-SCI. The SCI resulted in decreased mitochondrial protein expression, including PGC-1α, in the injury and peri-injury sites as early as 3 days post-injury. Formoterol treatment attenuated this decrease in PGC-1α, indicating enhanced MB, and restored downstream mitochondrial protein expression to that of controls by 15 days. Formoterol-treated mice also exhibited less histological damage than vehicle-treated mice 3 days after injury-namely, decreased lesion volume and increased white and gray matter sparing in regions rostral and caudal to the injury epicenter. Importantly, locomotor capability of formoterol-treated mice was greater than vehicle-treated mice by 7 days, reaching a Basso Mouse Scale score two points greater than that of vehicle-treated SCI mice by 15 days. Interestingly, similar locomotor restoration was observed when initiation of treatment was delayed until 8 h post-injury. These data provide evidence of ADRB2-mediated MB as a therapeutic approach for the management of SCI.

T-tubule remodelling disturbs localized ß2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure.

AIMS: Cardiomyocyte ß2-adrenergic receptor (ß2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors' subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), ß2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon ß2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown. METHODS AND RESULTS: Rat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), ß2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local ß2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the ß2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell ß2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve ß2AR-mediated signal compartmentation or reduce cAMP diffusion. CONCLUSION: Although changes in T-tubule structure and ß2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of ß2AR-mediated cAMP is temporarily reversed. Complete disorganization of ß2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

Silver nanoparticles stimulate glycogenolysis in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Silver nanoparticles (AgNPs) are found in many consumer products yet their biological effects on non-target aquatic organisms are yet to be fully understood. This research aimed to investigate the effects of AgNPs on cell signaling in rainbow trout (Oncorhynchus mykiss) hepatocytes. We focused on the ß-adrenoreceptor (AR), which mediates glycogenolysis, and the glucocorticoid receptor (GCR), which mediates gluconeogenesis. These two receptors have been extensively studied in trout hepatocytes due to their key roles during the stress response to increase glucose availability (among other things), allowing the organisms to cope with the stressor. We show for the first time that AgNPs at a concentration of 1 µg/mL did not interfere with the function of either the ß-AR or the GCR systems in rainbow trout hepatocytes, but at the concentration of 10 µg/mL AgNPs stimulated glycogenolysis which was apparently receptor-independent. This study suggests that AgNPs could affect hormone-regulated cell signaling pathways at a concentration of 10 µg/mL.