Nebivolol, a third generation β-adrenoceptor (β-AR) antagonist (β-blocker), causes vasodilation by inducing nitric oxide (NO) production. The mechanism via which nebivolol induces NO production remains unknown, resulting in the genesis of much of the controversy regarding the pharmacological action of nebivolol. Carvedilol is another β-blocker that induces NO production. A prominent pharmacological mechanism of carvedilol is biased agonism that is independent of Gαs and involves G protein-coupled receptor kinase (GRK)/β-arrestin signaling with downstream activation of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). Due to the pharmacological similarities between nebivolol and carvedilol, we hypothesized that nebivolol is also a GRK/β-arrestin biased agonist. We tested this hypothesis utilizing mouse embryonic fibroblasts (MEFs) that solely express β2-ARs, and HL-1 cardiac myocytes that express β1- and β2-ARs and no detectable β3-ARs. We confirmed previous reports that nebivolol does not significantly alter cAMP levels and thus is not a classical agonist. Moreover, in both cell types, nebivolol induced rapid internalization of β-ARs indicating that nebivolol is also not a classical β-blocker. Furthermore, nebivolol treatment resulted in a time-dependent phosphorylation of ERK that was indistinguishable from carvedilol and similar in duration, but not amplitude, to isoproterenol. Nebivolol-mediated phosphorylation of ERK was sensitive to propranolol (non-selective β-AR-blocker), AG1478 (EGFR inhibitor), indicating that the signaling emanates from β-ARs and involves the EGFR. Furthermore, in MEFs, nebivolol-mediated phosphorylation of ERK was sensitive to pharmacological inhibition of GRK2 as well as siRNA knockdown of β-arrestin 1/2. Additionally, nebivolol induced redistribution of β-arrestin 2 from a diffuse staining pattern into more intense punctate spots. We conclude that nebivolol is a β2-AR, and likely β1-AR, GRK/β-arrestin biased agonist, which suggests that some of the unique clinically beneficial effects of nebivolol may be due to biased agonism at β1- and/or β2-ARs.

Objectives Marrow adipose tissue may be modulated by physical activity and reambulation after inactivity. The aim of this study was to measure the effect of reambulation for up to 2 years after 60 days of bed rest on the lumbar bone marrow composition. Methods In a prospective clinical trial, 20 healthy men participated in a 60-day, 6-degree head-down tilt bed rest study. Serial 3-T magnetic resonance (MR) imaging measures of the lumbar spine were performed at baseline, after 57 days of bed rest, and at 30, 360, and 720 days of reambulation (100 MR imaging scans). Proton density with and without fat saturation, 2-point Dixon, and single-voxel MR spectroscopy techniques were used to assess bone marrow composition (300 measures). Erythropoiesis was measured using hematocrit, reticulocyte, and ferritin. Also, participants randomly received either a nutritional intervention composed of polyphenols, omega-3, vitamin E, and selenium or a normal diet. Results Thirty days of reambulation after 60 days of bed rest caused a marked decrease of the mean lumbar vertebral fat fraction (VFF) (−9.2 ± 1.6 percentage points, −8.0 ± 1.3 percentage points, and −12.7 ± 1.2 percentage points compared with baseline using proton density, Dixon, MR spectroscopy, respectively; all 3, P < 0.05). Reambulation also decreased the fat saturation index (−5.3 ± 1.1 percentage points compared with baseline; P < 0.05). These coincided with lower hematocrit and ferritin and with increased reticulocytes at reambulation day 13 compared with baseline (all 3, P < 0.05). After 57 days of bed rest, the VFF was unchanged from baseline (all 3 MR techniques, P > 0.05); reambulation for 2 years returned the lumbar VFF to baseline values. Interpretation This longitudinal trial established that 30 days of reambulation after 60 days of bed rest constituted a powerful stimulus for bone marrow reconversion. In this model, the enhanced erythropoiesis coupled with preferential consumption of fatty acids from regulated marrow adipose tissue to supply energy for erythropoiesis and bone anabolism may explain the lumbar vertebrae reconversion. These results will help interpreting bone marrow signal in ambulatory patients after long periods of bed rest.

G protein‐coupled receptor kinases 4γ (GRK4γ) is associated with hypertension. However, the molecular mechanism of action of GRK4γ remains largely unknown. From a proteomic examination of the binding partners of GRK4γ, we identified Gαs, Heat Shock Protein 90 (Hsp90) and an E3 ubiquitin ligase as potential binding partners. Additionally, previous studies suggested that GRK4γ is ubiquitinated. GRK2 and GRK5 are known to be ubiquitinated while SUMOylation, a small Ub‐like modifier protein that also attaches to lysine, has not been extensively studied in any GRKs. Therefore, we tested the hypothesis that GRK4γ can be ubiquitinated and SUMOylated. To demonstrate Ubiquitination and SUMOylation, we immunoprecipitated (IPed) GRK4γ and blotted for Ub or SUMO. The data clearly showed that GRK4γ is ubiquitinated but not SUMOylated. Interesting, GRK4γ ubiquitination is significantly enhanced after stimulation of MG132, a cell‐permeable proteasome inhibitor, but remains unchanged in the presence of two other proteasome inhibitors (Lactacystin and proteasome inhibitor I) which is counter to the previous studies. Further studies will be conducted to identify the role of GRK4γ activity in relation to GRK4γ ubiquination as well as identify the ubiquitination sites on GRK4γ and the role that ubiquitination plays in GRK4γ activity.

The angiotensin (Ang) II type 1 receptor (AT1R) is the target for a widely used class of drugs named angiotensin receptor blockers (ARBs). Polymorphisms in the ARB binding site are known to alter affinity. Mutagenesis studies identifying the binding site are limited as the site can be distorted simply by altering helical rotation/position. Previously, we demonstrated that wild‐type (wt) opossum (o) AT1R does not bind ARBs. Therefore, we made a model of the human (h) AT1R and oAT1R based on the structure of the mu‐opioid receptor through an iterative process. The models were optimized using all atomic molecular dynamics (MD) simulation with explicit membrane bilayer and water solvent. Utilizing docking and MD simulations the ARB binding pocket was elucidated; currently, both K199 in the 3rd helix and the backbone near the lipid‐solvent interface appear to be involved in binding. Additionally, we cloned the hAT1R and made individual point mutations reflecting the differences between hAT1R and oAT1R. Each receptor was stably transfected into HEK cells and characterized to determine if there are alterations in Ang II signaling as well as ARB‐mediated inhibition of signaling. Iterations of modeling and wet lab work will refine the ARB binding site allowing for identification of ARB resistant patients. Grant Funding Source: T. Taylor was supported by an APS Frontiers in Physiology Fellowship.

Space travel requires metabolic adaptations from multiple systems. While vital to bone and blood production, human bone marrow adipose (BMA) tissue modulation in space is unknown. Here we show significant downregulation of the lumbar vertebrae BMA in 14 astronauts, 41 days after landing from six months’ missions on the International Space Station. Spectral analyses indicated depletion of marrow adipose reserves. We then demonstrate enhanced erythropoiesis temporally related to low BMA. Next, we demonstrated systemic and then, local lumbar vertebrae bone anabolism temporally related to low BMA. These support the hypothesis that BMA is a preferential local energy source supplying the hypermetabolic bone marrow postflight, leading to its downregulation. A late postflight upregulation abolished the lower BMA of female astronauts and BMA modulation amplitude was higher in younger astronauts. The study design in the extreme environment of space can limit these conclusions. BMA modulation in astronauts can help explain observations on Earth.