Description: Salmeterol is a long-acting β 2-adrenergic receptor ( β 2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the β 2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein–coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded- β 2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to β 2AR relative to isoproterenol. The ratio of BRET ISO /BRET SALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRET ISO and a stable BRET SALM . Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0–30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein–coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A–mediated events.

Description: Designer receptors exclusively activated by a designer drug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediated versus β-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3 muscarinic receptor that can activate Gq/11 with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is active in vivo and that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependent versus β-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.

Description: Adipose tissue constitutes an extremely active endocrine organ with a network of signaling pathways enabling the organism to adapt to a wide range of different metabolic challenges, such as starvation, stress, infection, and short periods of gross energy excess. The functional pleiotropism of adipose tissue relies on its ability to synthesize and release a huge variety of hormones, cytokines, complement and growth factors, extracellular matrix proteins, and vasoactive factors, collectively termed adipokines. Obesity is associated with adipose tissue dysfunction leading to the onset of several pathologies including type 2 diabetes, dyslipidemia, nonalcoholic fatty liver, or hypertension, among others. The mechanisms underlying the development of obesity and its associated comorbidities include the hypertrophy and/or hyperplasia of adipocytes, adipose tissue inflammation, impaired extracellular matrix remodeling, and fibrosis together with an altered secretion of adipokines. Recently, the potential role of brown and beige adipose tissue in the protection against obesity has been also recognized. In contrast to white adipocytes, which store energy in the form of fat, brown and beige fat cells display energy-dissipating capacity through the promotion of triacylglycerol clearance, glucose disposal, and generation of heat for thermogenesis. Identification of the morphological and molecular changes in white, beige, and brown adipose tissue during weight gain is of utmost relevance for the identification of pharmacological targets for the treatment of obesity and its associated metabolic diseases.

Description: The grapsid crab Chasmagnathus granulata populates brackish-water lagoons and other estuarine environments. In its reproduction, this species follows a strategy of larval export, i.e. its larvae live under different salinity conditions from the juveniles and adults. In the present experimental investigation, ontogenetic changes in the capability for osmoregulation were studied in all 4 zoeal stages, the megalopa, the juvenile crab instars I, II and IV, and adults (all reared in seawater, 32). Moreover, we studied effects of embryonic and larval acclimation on osmoregulation. The zoea I larvae were slight hyper-regulators at low salinities (10 to 17) and hyper-osmoconformers at higher salinities. Stages II to IV zoe generally hyper-osmoconformers. At metamorphosis to the megalopa, the type of osmoregulation changed to hyper-hypo-regulation. The osmoregulatory capacities under both hypo- and hypersaline conditions increased strongly in the crab I and throughout later juvenile development. These patterns in osmoregulation match the ontogenetic changes that typically occur in the ecology of C. granulata: the zoea I hatches in brackish estuarine waters, where the juveniles and adults live, before it is exported to coastal marine zones. This initial larval stage is euryhaline and capable of hyper-osmoregulation at low salinities. The same capabilities were observed in the megalopa, which re-invades the brackish adult environment. This stage is known to settle in semiterrestrial habitats near the adult burrows, where both brackish and hypersaline conditions are likely to occur; this coincides with the first ontogenetic appearance of the hyper-hypo-osmoregulation pattern. The zoeal stages II, III and IV, in contrast, develop in the adjacent sea, where the salinity is higher and more stable. Correspondingly, these intermediate larval stages were found to be stenohaline osmoconformers. Preceding exposure of the eggs and larvae to a reduced salinity (20) enhanced the hyper-osmoregulatory capacity at low salinities (5 to 10) in all zoeal stages. This indicates an effect of non-genetic acclimation and, hence, phenotypic plasticity. This trait should have an adaptive value, as it increases the chance larval survival, at least in the initial larval stage, which is in the field exposed to highly variable, mostly reduced salinities.

Description: Spatial distribution of macrofaunaalong the longshore axis was studied on an exposed (Uruguayan) sandybeach in order to assess (1) its dependence on beach topography,and (2) the validity to extrapolate local distribution patterns to largerspatial scales (i.e. km). The distribution of the isopods Excirolanaarmata and Excirolana braziliensis, the sand crab Emeritabrasiliensis and several species of insects was analyzed at 3spatial scales. A grid sampling was used at small scale(intersample distance: 4 m; extension sampled: 30 to 40 m), atransect design at meso scale (intersample distance 4 to 20 m;extension sampled: 100 to 120 m), and random sampling at largescale (intersample distance: 100 m; extension sampled: 3000 m).Spatial distribution at small and meso scales were describedusing autocorrelation functions, and tested for effects oftopography at meso and large scales using ANOVA and pairedt-tests. We found that at small and meso scales the distribution ofE. armata and E. brasiliensis was patchy and affected by cusptopography. At large scales, the effect of cusp topography wasrestricted to E. armata. E. armata and E. brasiliensis showedlarge-scale aggregations, and E. braziliensis and insects showedlarge-scale patchiness associated with longshore variability insediment water content and dune characteristics. We conclude thatcusp topography affects longshore distribution patterns, and that itis not valid to extrapolate local longshore distribution to largerscales for every species. We suggest that different processesaffecting spatial distribution must be operating at different scales.At small scales, patterns of distribution may be affected by swashtransport of food and/or organisms, involving factors such as beachtopography, swash and animal movements. At larger scales, inaddition to larval and food supply, sediment transport by the windwould play an important role. Spatial patterns of sand transport bythe wind may be affected by longshore changes in vegetationcover on the dune field. We further suggest that there may be a linkbetween the beach and the dune habitats operating at large spatialscales.

Description: We studied interrelationships between initial egg size and biomass, duration of embryogenesis at different salinities, and initial larval biomass in an estuarine crab, Chasmagnathus granulata. Ovigerous females were maintained at three different salinities (15, 20 and 32); initial egg size (mean diameter), biomass (dry weight, carbon and nitrogen) as well as changes in egg size, embryonic development duration, and initial larval biomass were measured. Initial egg size varied significantly among broods from different females maintained under identical environmental conditions. Eggs from females maintained at 15 had on average higher biomass and larger diameter. We hypothesise that this is a plastic response to salinity, which may have an adaptive value, i.e. it may increase the survivorship during postembryonic development. The degree of change in egg diameter during the embryonic development depended on salinity: eggs in a late developmental stage were at 15 significantly larger and had smaller increment than those incubated at higher salinities. Development duration was longer at 15, but this was significant only for the intermediate embryonic stages. Initial larval biomass depended on initial egg size and on biomass loss during embryogenesis. Larvae with high initial biomass originated either from those eggs that had, already from egg laying, a high initial biomass (reflecting individual variability under identical conditions), or from those developing at a high salinity (32), where embryonic biomass losses were generally minimum. Our results show that both individual variability in the provisioning of eggs with yolk and the salinity prevailing during the embryonic development are important factors causing variability in the initial larval biomass of C. granulata, and thus, in early larval survival and growth.

Description: Activity rhythms of two cirolanid isopods, Excirolana armata and Excirolana braziliensis, were studiedbased on both seasonal field observations and laboratory experiments, at an exposed microtidal sandy beach inUruguay. The natural emergence patterns of both species were observed in the field for 1 year, twice in each season,and correlated to sea level, expected tidal cycles and diel cycles. Laboratory experiments were carried out in order todetect endogenous rhythms of activity and observe how emergence of both species was affected by changes in lightand/or sediment thixotropy. We also compared behavioral strategies of sympatric species that occupy different beachlevels. Sea level (and thus swash zone position) during field sampling did not follow expected tidal cycles for mostsampling occasions. E. armata was observed in activity most of the time, but activity only correlated with sea level onthree out of eight occasions, and only once was correlated to expected tidal cycle. Laboratory results showed thatemergence under constant conditions was rare; changes in sediment thixotropy stimulated emergence, but theresponse was not cyclical; light had little effect on this response. On the other hand, E. braziliensis was fairly scarce inthe water column, but swimming individuals were observed always during the night. They displayed an endogenouscircadian activity pattern in the laboratory which augmented in response to changes in sediment thixotropy. The naturallight/dark cycle modulated both spontaneous and response emergence by increasing day/night differences in activity.In this study E. armata, a midlittoral species more exposed to sea level variations, seemed to rely entirely on differentphysical and/or biological cues to trigger emergence at the appropriate time. E. braziliensis, found mostly in the upperintertidal zone, emerged in a circadian rhythm, which was stimulated by changes in sediment thixotropy and reinforcedby light cycles. The results of this study led us to conclude that on microtidal, unpredictable beaches, local physicaland biological factors can combine to determine different activity strategies in organisms from different intertidal levels.

Description: Present theory of marine community structure includes the effect of environmental factors that act upon larval survival on distribution and abundance of benthic adults. The theory assumes that pre-settlement environmental factors produce a direct effect at the population level, determining the abundance of individuals that enter the benthic habitat. These are density effects that propagate from the larval to the benthic phase (abbreviated here as DEMPHs). However, recent information suggests that pre-settlement factors may also produce an effect on population abundance, mediated by changes in traits of settlers. For instance, larvae experiencing food stress may give rise to juveniles with reduced energy reserves, growth rate, and in consequence with a low life expectancy. Thus, such trait-mediated (e.g. life historical, physiological) effects propagate through different life phases of the complex life cycles of benthic invertebrates, ultimately affecting juvenile survival. In this paper, I discuss the potential role of this special kind of trait-mediated effect (abbreviated here as DELPHIs) on benthic communities. I show that DELPHIs may affect the shape of the stock-settlement function leading to an erroneous interpretation of the role of post-settlement factors on benthic communities. Mortality due to DELPHIs is the consequence of pre-settlement factors but may be confounded with mortality due to post-settlement factors. In this regard, I conclude that the only solution to avoid this error is to incorporate measurements of traits of individuals in settlement and recruitment studies. Besides, DELPHIs may allow for complex interactions between pre- and post-settlement processes, suggesting that dichotomous views about the role of pre- and post-settlement processes are not appropriate. Finally, I suggest that future field research in benthic communities should incorporate an estimation of DELPHIs as a tool to expand, and thus improve, marine community theory.