Background and purpose: The CB 2 receptor has been proposed as a novel target for the treatment of pain, and CB 2 receptor agonists defined in in vitro assays have demonstrated analgesic activity in animal models. Based on its in vivo analgesic efficacy, AM1241 has been classified as a CB 2 -selective agonist. However, in vitro characterization of AM1241 in functional assays has not been reported. Experimental approach: In this study, AM1241 was characterized across multiple in vitro assays employing heterologous recombinant receptor expression systems to assess its binding potencies at the human CB 2 and CB 1 receptors and its functional efficacies at the human CB 2 receptor. Key results: AM1241 exhibited distinct functional properties depending on the assay conditions employed, a unique profile in contrast to those of the agonist CP 55,940 and the inverse agonist SR144528. AM1241 displayed neutral antagonist activities in FLIPR and cyclase assays. However, when cyclase assays were performed using lower forskolin concentrations for stimulation, AM1241 exhibited partial agonist efficacy. In addition, it behaved as a partial agonist in ERK (or MAP) kinase assays. Conclusions and implications:The unusual phenomenon of inconsistent functional efficacies suggests that AM1241 is a protean agonist at the CB 2 receptor. We postulate that functional efficacies displayed by protean agonists in various assay systems may depend on the levels of receptor constitutive activities exhibited in the assay systems, and therefore, efficacies observed in in vitro assays may not predict in vivo activities.

Gavini CK, Mukherjee S, Shukla C, Britton SL, Koch LG, Shi H, Novak CM. Leanness and heightened nonresting energy expenditure: role of skeletal muscle activity thermogenesis. Am J Physiol Endocrinol Metab 306: E635-E647, 2014. First published January 7, 2014; doi:10.1152/ajpendo.00555.2013.-A high-calorie diet accompanied by low levels of physical activity (PA) accounts for the widespread prevalence of obesity today, and yet some people remain lean even in this obesogenic environment. Here, we investigate the cause for this exception. A key trait that predicts high PA in both humans and laboratory rodents is intrinsic aerobic capacity. Rats artificially selected as high-capacity runners (HCR) are lean and consistently more physically active than their low-capacity runner (LCR) counterparts; this applies to both males and females. Here, we demonstrate that HCR show heightened total energy expenditure (TEE) and hypothesize that this is due to higher nonresting energy expenditure (NREE; includes activity EE). After matching for body weight and lean mass, female HCR consistently had heightened nonresting EE, but not resting EE, compared with female LCR. Because of the dominant role of skeletal muscle in nonresting EE, we examined muscle energy use. We found that lean female HCR had higher muscle heat dissipation during activity, explaining their low economy of activity and high activity EE. This may be due to the amplified skeletal muscle expression levels of proteins involved in EE and reduced expression levels of proteins involved in energy conservation in HCR relative to LCR. This is also associated with an increased sympathetic drive to skeletal muscle in HCR compared with LCR. We find little support for the hypothesis that resting metabolic rate is correlated with maximal aerobic capacity if body size and composition are fully considered; rather, the critical factor appears to be activity thermogenesis.nonexercise activity thermogenesis; high-and low-capacity runners; energy expenditure; obesity OBESITY IS INCREASING WORLDWIDE and underlies innumerable associated health problems that lead to decreased quality of life and increased mortality (3,19,20,25,28,75). Although the contribution of diet to the obesogenic environment is unquestioned (69), a sedentary lifestyle also has deleterious effects on body weight (BW) and health (8,15,33). An individual's level of daily physical activity (PA) is a biologically regulated heritable trait that tends to be associated with leanness (31,47,48). We have found that a key feature predicting high PA is intrinsic aerobic capacity; this holds true for both humans and laboratory rodents (35,37,39,56,63,96). Rats that have been selectively bred for high aerobic endurance treadmill running capacity [high-capacity runners (HCR)] are consistently more physically active than their low-endurance counterparts selectively bred as low-capacity runners (LCR). Aerobic capacity is a strong predictor of early morbidity and mortality in both men and women (39, 56); in fact, after age, it is ...

Classically, Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in ERK activation following G-protein–coupled receptor (GPCR) activation. Here we show that PI3Kγ noncanonically regulates ERK phosphorylation in a kinase-independent mechanism, irrespective of the upstream signals. PI3Kγ sequesters PP2A, allowing sustained ERK function.

Purpose:We sought to determine microbe-metabolite composition and interactions within indwelling ureteral stent biofilms, determine their association with patient factors including infection, and reconstitute biofilm formation on relevant surface materials in vitro.Materials and Methods:Upon ureteral stent removal from patients, proximal and distal ends were swabbed. Samples were analyzed by 16S next-generation sequencing and metabolomics. A continuous-flow stir-tank bioreactor was used to reconstitute and quantify in vitro biofilm formation from stent-isolated bacteria on stent-related materials including silicone, polytetrafluoroethylene, polyurethane, polycarbonate, and titanium. Diversity, relative abundance, and association with clinical factors were analyzed with ANOVA and Bonferroni t-tests or PERMANOVA. Biofilm deposition by microbial strain and device material type were analyzed using plate counts and scanning electron microscopy following bioreactor incubation.Results:All 73 samples from 37 ureteral stents harbored microbiota. Specific genera were more abundant in samples from stents wherein there was antibiotic exposure during indwelling time (Escherichia/Shigella, Pseudomonas, Staphylococcus, Ureaplasma) and in those associated with infection (Escherichia/Shigella, Ureaplasma). The enriched interaction subnetwork in stent-associated infection included Ureaplasma and metabolite 9-methyl-7-bromoeudistomin. Strains identified as clinically relevant and central to interaction networks all reconstituted biofilm in vitro, with differential formation by strain (Enterococcus faecalis most) and material type (titanium least).Conclusions:Ureteral stent biofilms exhibit patterns unique to stent-associated infection and antibiotic exposure during indwelling time. Microbes isolated from stents reconstituted biofilm formation in vitro. This work provides a platform to test novel materials, evaluate new coatings for anti-biofilm properties, and explore commensal strain use for bacterial interference against pathogens.

Dysregulation of immune responses has been linked to the generation of IgG autoantibodies that target human β1ARs and contribute to deleterious cardiac outcomes. Given the benefits of β-blockers observed in patients harboring IgG3 sub-class of autoantibodies, we investigated the role of these autoantibodies in human β1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using HEK293 cells expressing human β1ARs. Unexpectedly, pre-treatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylyl cyclase (AC) activity and cAMP generation while enhancing biased β-arrestin recruitment and ERK activation. Contrastingly, the β-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Since IgG3(+) autoantibodies are specific to human β1ARs, non-failing human hearts were used as an endogenous system to determine their ability to bias β1AR signaling. Consistently, metoprolol increased AC activity reflecting the ability of the IgG3(+) autoantibodies to bias β-blocker towards G-protein coupling. Importantly, IgG3(+) autoantibodies are specific towards β1AR as they did not alter β2AR signaling. Thus, IgG3(+) autoantibody biases β-blocker towards G-protein coupling, while impairing agonist-mediated G-protein activation but promoting G-protein-independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) β1AR autoantibodies.