Characterization of Rimonabant effects on G protein activity

G protein-coupled receptor (GPCRs) is the largest class of cell-surface receptors, and represents today the target of 40% of the drugs in the pharmaceutical market. In the absence of agonists, many GPCRs have found to exhibit spontaneous activity, which can be blocked by ligands that are referred to as inverse agonists (Milligan, 2003). Cannabinoid CB1 receptor is one of the most abundant GPCR in the central nervous system, and is coupled to Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinase (MAPK), inhibit voltage gated Ca2+ channels and activate inwardly rectifying K+ channels (Howlett et al., 2000; Pertwee, 2010). The first selective and potent CB1 antagonist Rimonabant (also known SR141716A, SR) at high micromolar concentrations behaves as an inverse agonist, i.e. decreases [35S]GTPγS binding in rodent and human cerebral cortex and in Chinese hamster ovary (CHO) cells transfected with CB1 receptors (Rinaldi-Carmona, 1994). However, in vitro and in vivo studies performed using CB1 knockout (KO) and CHO cells not expressing CB1 receptors suggest that inverse agonist activity of SR is CB1 receptor independent (Pertwee, 2005). Several hypotheses have been postulated to explain the inverse agonism of SR, including its action on different receptors (i.e GPCR mainly coupled to Gi/o proteins) and/or its negative modulation of the constitutive activity of CB1 receptors. Alternatively, SR might explicate these “inverse agonist effect” in a manner receptor-independent acting directly on G protein level. The present study aimed to determine whether the CB1 receptor-independent effects of SR are mediated via GPCRs, in particular GABAB and dopamine D2 receptors, that share the same Gαi/o signaling pathways, or if SR acts directly on G protein subunits. For this purpose we investigated the molecular mechanisms of SR on G protein activity in native and recombinant systems by using different experimental approaches (i.e., GTPγS binding, bioluminescence Resonance Energy Transfer (BRET), electrophysiological recordings). In particular, we first evaluated the effects of SR on basal and agonist-stimulated [35S]GTPγS binding in systems containing CB1, GABAB and D2 receptor populations (i.e., rat membrane homogenates and CHO stable transfected with GABAB or D2 receptors), and in systems lacking CB1 and GABAB receptors (i.e., CB1- and GABAB-KO mice). Then, using BRET approach we monitored dissociation between Gαo and Gβγ subunits and their conformational rearrangements before and after GABAB receptors activation. In addition, using the same assay we studied the molecular interaction between D2 receptor and Gαi1 protein subunits (Gαi1-60, Gαi1-91 and Gαi1-121). Next, we evaluated the effects of SR on adenylate cyclase activity, using BRET with the CAMYEL sensor, a recent technique developed to detect the level of cAMP in living cells. Specifically, the inhibitory effect of SR on Gi and Gs protein pathways measuring BRET signal in cells transfected with CAMYEL and GABAB, D2 or D1 receptors was investigated. Finally, whole cell voltage clamp recordings from midbrain dopamine neurons in acute rat brain slices ex vivo were performed to evaluate the effects of SR on baclofen and quinpirole-induced outward K+ current both in wild-type (WT) and CB1-KO mice. In addition, in order to demonstrate that SR induced the inhibition of GIRK channel activity by acting directly on G protein, we use a GPCR-free experimental setup (i.e. whole cell patch clamp experiments were performed in CHO cells transfected with GIRK1/2). The main finding of this study is that SR, at micromolar concentrations, prevented GPCR-G protein signaling through a direct interaction with the G proteins mainly with the subunits αi/o

Characterization of Rimonabant effects on G protein activity

G protein-coupled receptor (GPCRs) is the largest class of cell-surface receptors, and represents today the target of 40% of the drugs in the pharmaceutical market. In the absence of agonists, many GPCRs have found to exhibit spontaneous activity, which can be blocked by ligands that are referred to as inverse agonists (Milligan, 2003). Cannabinoid CB1 receptor is one of the most abundant GPCR in the central nervous system, and is coupled to Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinase (MAPK), inhibit voltage gated Ca2+ channels and activate inwardly rectifying K+ channels (Howlett et al., 2000; Pertwee, 2010). The first selective and potent CB1 antagonist Rimonabant (also known SR141716A, SR) at high micromolar concentrations behaves as an inverse agonist, i.e. decreases [35S]GTPγS binding in rodent and human cerebral cortex and in Chinese hamster ovary (CHO) cells transfected with CB1 receptors (Rinaldi-Carmona, 1994). However, in vitro and in vivo studies performed using CB1 knockout (KO) and CHO cells not expressing CB1 receptors suggest that inverse agonist activity of SR is CB1 receptor independent (Pertwee, 2005). Several hypotheses have been postulated to explain the inverse agonism of SR, including its action on different receptors (i.e GPCR mainly coupled to Gi/o proteins) and/or its negative modulation of the constitutive activity of CB1 receptors. Alternatively, SR might explicate these “inverse agonist effect” in a manner receptor-independent acting directly on G protein level. The present study aimed to determine whether the CB1 receptor-independent effects of SR are mediated via GPCRs, in particular GABAB and dopamine D2 receptors, that share the same Gαi/o signaling pathways, or if SR acts directly on G protein subunits. For this purpose we investigated the molecular mechanisms of SR on G protein activity in native and recombinant systems by using different experimental approaches (i.e., GTPγS binding, bioluminescence Resonance Energy Transfer (BRET), electrophysiological recordings). In particular, we first evaluated the effects of SR on basal and agonist-stimulated [35S]GTPγS binding in systems containing CB1, GABAB and D2 receptor populations (i.e., rat membrane homogenates and CHO stable transfected with GABAB or D2 receptors), and in systems lacking CB1 and GABAB receptors (i.e., CB1- and GABAB-KO mice). Then, using BRET approach we monitored dissociation between Gαo and Gβγ subunits and their conformational rearrangements before and after GABAB receptors activation. In addition, using the same assay we studied the molecular interaction between D2 receptor and Gαi1 protein subunits (Gαi1-60, Gαi1-91 and Gαi1-121). Next, we evaluated the effects of SR on adenylate cyclase activity, using BRET with the CAMYEL sensor, a recent technique developed to detect the level of cAMP in living cells. Specifically, the inhibitory effect of SR on Gi and Gs protein pathways measuring BRET signal in cells transfected with CAMYEL and GABAB, D2 or D1 receptors was investigated. Finally, whole cell voltage clamp recordings from midbrain dopamine neurons in acute rat brain slices ex vivo were performed to evaluate the effects of SR on baclofen and quinpirole-induced outward K+ current both in wild-type (WT) and CB1-KO mice. In addition, in order to demonstrate that SR induced the inhibition of GIRK channel activity by acting directly on G protein, we use a GPCR-free experimental setup (i.e. whole cell patch clamp experiments were performed in CHO cells transfected with GIRK1/2). The main finding of this study is that SR, at micromolar concentrations, prevented GPCR-G protein signaling through a direct interaction with the G proteins mainly with the subunits αi/o

A new system of authorship best assessment

Purpose:The standard bibliometric indexes ("m-quotient "H-," "H2-," "g-," "a-," "m-," and "r-" index) do not considered the research' position in the author list of the paper. We proposed a new methodology, System of Authorship Best Assessment (SABA), to characterize the scientific output based on authors' position. Material and Methods:Four classes S1A, S1B, S2A, and S2B include only papers where the researcher is in first, first/last, first/second/last, and first/second/second-last/last position respectively were used for the calculation of H-index and number of citations The system was tested with Noble prize winners controlled with researchers matched for H-index. The different in percentage between standard bibliometric index and S2B was calculated and compared. Results:The percentage differences in Noble prize winners between S2B-H-index versus Global H-index and number of citations is very lower comparing with control group (median 4.15% [adjusted 95% CI, 2.54-5.30] vs 9.00 [adjusted 95% CI, 7.16-11.84], p < 0.001; average difference 8.7% vs 20.3%). All different in percentage between standard bibliometric index and S2B except two (H2- and m-index) were significantly lower among Noble prize compared with control group. Conclusion:The SABA methodology better weight the research impact by showing that for excellent profiles the S2B is similar to global values whereas for other researchers there is a significant difference

Vaccination attitude and communication in early settings: An exploratory study

Background: This study assesses attitudes towards vaccination in mothers of new-born babies and explores its association with different exposures to communication. Methods: Data were collected through questionnaires administered by means of interviews. Results: Data highlighted that 20% of mothers showed an orientation towards vaccine hesitancy. As for the reasons behind the attitude to vaccine hesitancy, data showed that concern is a common feature. As for the different exposures to communication, 49% of mothers did not remember having received or looked for any information about vaccination during pregnancy and post-partum; 25% stated they received information from several healthcare and non-healthcare sources; 26% declared having received or looked for information by means of healthcare and non-healthcare sources, as well as having taken part in a specific meeting during antenatal classes or at birth centres. The attitude towards vaccine hesitancy tends to reduce as exposure to different communication increases. Conclusions: This study supports the hypothesis that participation in interactive meetings in small groups focused on vaccination during the prenatal course or at the birth point may act as an enabling factor contributing to a decrease in the tendency to experience vaccine hesitation

Failure of Acute Ethanol Administration to Alter Cerebrocortical and Hippocampal Allopregnanolone Levels in C57BL/6J and DBA/2J Mice

Ethanol (EtOH) administration increases brain allopregnanolone levels in rats, and this increase contributes to sensitivity to EtOH's behavioral effects. However, EtOH's effects on allopregnanolone may differ across species. We investigated the effects of acute EtOH administration on allopregnanolone, progesterone, and corticosterone levels in cerebral cortex and hippocampus of C57BL/6J and DBA/2J mice, 2 inbred strains with different alcohol sensitivity

Chronic treatment with hormonal contraceptives alters hippocampal BDNF and histone H3 post-translational modifications but not learning and memory in female rats

Hormonal contraceptives prevent ovulation with subsequent reduction in endogenous levels of estradiol, progesterone and its neuroactive metabolite allopregnanolone. These neurosteroids modulate several brain functions, including neuronal plasticity, cognition and memory. We hypothesized that hormonal contraceptives might affect synaptic plasticity, learning and memory, as a consequence of suppressed endogenous hormones levels. Female rats were orally treated with a combination of ethinyl estradiol (EE, 0.020 mg) and levonorgestrel (LNG, 0.060 mg) once daily for four weeks. Decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and altered histone H3 post-translational modifications (PTMs) were observed 14 days after discontinuation from chronic EE-LNG treatment. These effects were not accompanied by alterations in long-term plasticity at glutamatergic synapses, recognition memory in the novel object and novel place location tests, or spatial learning, memory, and behavioral flexibility in the Morris water maze test. Thus, decreased BDNF content does not affect synaptic plasticity and cognitive performance; rather it might be relevant for the occurrence of certain psychiatric symptoms, reported by some women using hormonal contraceptives. These results provide the first evidence of hippocampal epigenetic changes induced by hormonal contraceptives and complement previous studies on the neurobiological actions of hormonal contraceptives; the finding that effects of chronic EE-LNG treatment on BDNF content and histone PTMs are observed 14 days after drug discontinuation warrants further investigation to better understand the implications of such long-term consequences for women's health

The Use of Reproductive Indicators for Conservation Purposes: The Case Study of Palinurus elephas in Two Fully Protected Areas and Their Surrounding Zones (Central-Western Mediterranean)

Simple Summary The European spiny lobster Palinurus elephas is a species with a high commercial value that inhabits the Mediterranean Sea and the adjacent Atlantic waters and is classified by IUCN as vulnerable due to its continuous overfishing. In this study, we analyse the reproductive parameters of P. elephas populations in two different fully protected areas, including their surrounding commercial zones, in Sardinia (Italy, central-western Mediterranean), where a restocking programme was carried out. Here, data on fecundity, size at maturity, vitellogenin concentration and temporal trends of egg production of P. elephas was provided, and the relevance of this information for fisheries management and conservation planning is discussed. In 1990s, the European spiny lobster Palinurus elephas, one of the most commercially important species in the Mediterranean, exhibited a population decline. For this reason, fully protected areas (FPAs) appeared effective in re-establishing natural populations and supporting fishery-management objectives. Here, the reproductive parameters of P. elephas populations in two different FPAs (Su Pallosu and Buggerru, central-western Mediterranean), where a restocking programme was carried out, and in their surrounding commercial zones, were investigated from quantitative and qualitative perspectives. The comparison of fecundity between females collected inside and outside FPAs did not show statistical differences as well as the vitellogenin concentration, which did not vary among eggs of different size classes of females caught inside and outside the FPAs, indicating the same reproductive potential. The study demonstrated a benefit of overexploited populations in terms of enhancement of egg production overtime (15 years for Su Pallosu and 6 years for Buggerru) with a mean egg production 4.25-5.5 times higher at the end of the study than that observed at the beginning of the study. The main driver of eggs production appeared to be size, with larger lobsters more present inside the FPAs than outside. Given these results, the dominant contribution of the two studied FPAs to the regional lobster reproduction is remarkable

Predictors of long-term response to abiraterone in patients with metastastic castration-resistant prostate cancer: a retrospective cohort study

We aimed to identify clinical predictors of long-term response to abiraterone (defined as >12 months drug exposure) in a retrospective cohort of metastatic castration-resistant prostate cancer patients treated in post-docetaxel setting at 24 Italian centers. The Cox proportional hazards model was used to analyze the association between clinical features and the duration of drug exposure. Results were expressed as hazard ratios (HR) with associated 95% confidence intervals (CI). A total of 143 patients met the inclusion criteria. Their median age was 73 years, median Gleason score 8 and median abiraterone exposure 20 months. At the univariate analysis, a significant correlation with the duration of abiraterone exposure was found for Gleason score (HR 0.82, 95% CI 0.71-0.96; p=0.012), PSA (HR 1.10, 95% CI 1.03-1.18; p=0.08) and lactic dehydrogenase levels (HR 1.22, 95% CI 1.02-1.46; p=0.027), while the association between lower alkaline phosphatase levels and treatment duration was marginally significant (HR 1.07, 95% CI 0.99-1.16; p=0.074). Only PSA and Gleason score were predictive of long-term treatment duration in the multivariate analysis. No other clinical factors resulted to be predictive of sustained response to abiraterone, including metastatic disease at diagnosis and visceral disease, suggesting that all subgroups of patients may derive a substantial clinical benefit from abiraterone treatment. These findings need to be validated in prospective, larger studies