Functionally selective activation of the dopamine receptor D2 is mirrored by the protein expression profiles.

The development of functionally selective or biased ligands is a promising approach towards drugs with less side effects. Biased ligands for G protein-coupled receptors can selectively induce G protein activation or ß-arrestin recruitment. The consequences of this selective action on cellular functions, however, are not fully understood. Here, we investigated the impact of five biased and balanced dopamine D2 receptor agonists and antagonists on the global protein expression in HEK293T cells by untargeted nanoscale liquid chromatography-tandem mass spectrometry. The proteome analysis detected 5290 protein groups. Hierarchical clustering and principal component analysis based on the expression levels of 1462 differential proteins led to a separation of antagonists and balanced agonist from the control treatment, while the biased ligands demonstrated larger similarities to the control. Functional analysis of affected proteins revealed that the antagonists haloperidol and sulpiride regulated exocytosis and peroxisome function. The balanced agonist quinpirole, but not the functionally selective agonists induced a downregulation of proteins involved in synaptic signaling. The ß-arrestin-preferring agonist BM138, however, regulated several proteins related to neuron function and the dopamine receptor-mediated signaling pathway itself. The G protein-selective partial agonist MS308 influenced rather broad functional terms such as DNA processing and mitochondrial translation.

Rapid assessment of G protein signaling of four opioid receptors using a real-time fluorescence-based membrane potential assay.

Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.

Potentiation of P2X3 receptor mediated currents by endothelin-1 in rat dorsal root ganglion neurons.

Endothelin-1 (ET-1), an endogenous vasoconstrictor, has been known as a pro-nociceptive agent involved in multitude of pain. ET-1 acts on endothelin receptors on vascular endothelial cells, sensitizes release of ATP, which then acts on P2X3 receptors on nociceptors and results in mechanical hyperalgesia. Both endothelin receptors and P2X3 receptors are present in primary sensory neuron, where it remains unclear whether there is an interaction between them. Herein, we reported that ET-1 potentiated the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. ET-1 concentration-dependently increased α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents, which were mediated by P2X3 receptors. ET-1 shifted the α,ß-meATP concentration-response curve upwards, with an increase of 34.38 ± 4.72% in the maximal current response to α,ß-meATP in the presence of ET-1. ET-1 potentiation of α,ß-meATP-evoked currents was voltage-independent. ET-1 potentiated P2X3 receptor-mediated currents through endothelin-A receptors (ETAR), but not endothelin-B receptors (ETBR). ET-1 potentiation was supressed by blockade of intracellular G-protein or protein kinase C (PKC) signaling. Moreover, there is a synergistic effect on mechanical allodynia induced by intraplantar injection of ET-1 and α,ß-meATP in rats. Pharmacological blockade of P2X3 receptors also alleviated ET-1-induced mechanical allodynia. These results suggested that ET-1 sensitized P2X3 receptors in primary sensory neurons via an ETAR and PKC signaling pathway. Our data provide evidence that cutaneous ET-1 induced mechanical allodynia not only by increasing the release of ATP from vascular endothelial cells, but also by sensitizing P2X3 receptors on nociceptive DRG neurons.

Celiac disease serology and gut microbiome following proton pump inhibitor treatment.

BACKGROUND: Celiac disease is an autoimmune enteropathy characterized by an aberrant immune response to ingested gluten in genetically predisposed individuals. Studies have pointed to a rising prevalence of celiac disease in recent decades. Changes in diet and use of medication that may impact the gut microbiome have been suggested as potential contributors. Exposure to proton pump inhibitors (PPIs) was recently found to be associated with an increased risk for subsequent diagnosis of celiac disease. We aimed to investigate potential mechanisms for this link by examining the relationship between PPI use and gluten-related immune responses in the context of changes in gut microbiome. METHODS: We performed a post hoc analysis of blood and fecal samples from a recent randomized trial in order to assess the potential association between PPI use and development of celiac disease serology in conjunction with alterations in gastrointestinal microbial composition. The study included 12 healthy participants who were administered a PPI (Omeprazole; 40 mg twice daily) for 4 or 8 weeks. RESULTS: The analysis did not reveal an overall significant change in levels of serologic markers of celiac disease for the study cohort in response to PPI treatment. However, one individual developed a marked increase in the celiac disease-specific autoantibody response to transglutaminase 2 in conjunction with enhanced immune reactivity to gluten during the trial. Genotyping revealed positivity for the celiac disease-associated HLA-DQ2 and -DQ8 alleles. Furthermore, the observed elevation in antibody responses was closely associated with a sharp increase in fecal abundance of bacteria of the order Actinomycetales. CONCLUSIONS: The results of this exploratory analysis support further investigation of molecular mechanisms involved in the contribution of PPIs to celiac disease risk through the potential enhancement of gluten immunopathology and changes in gut microbial population.

Regulators of G protein signalling as pharmacological targets for the treatment of neuropathic pain.

Neuropathic pain, a specific type of chronic pain resulting from persistent nervous tissue lesions, is a debilitating condition that affects about 7% of the population. This condition remains particularly difficult to treat because of the poor understanding of its underlying mechanisms. Drugs currently used to alleviate this chronic pain syndrome are of limited benefit due to their lack of efficacy and the elevated risk of side effects, especially after a prolonged period of treatment. Although drugs targeting G protein-coupled receptors (GPCR) also have several limitations, such as progressive loss of efficacy due to receptor desensitization or unavoidable side effects due to wide receptor distribution, the identification of several molecular partners that contribute to the fine-tuning of receptor activity has raised new opportunities for the development of alternative therapeutic approaches. Regulators of G protein signalling (RGS) act intracellularly by influencing the coupling process and activity of G proteins, and are amongst the best-characterized physiological modulators of GPCR. Changes in RGS expression have been documented in a range of models of neuropathic pain, or after prolonged treatment with diverse analgesics, and could participate in altered pain processing as well as impaired physiological or pharmacological control of nociceptive signals. The present review summarizes the experimental data that implicates RGS in the development of pain with focus on the pathological mechanisms of neuropathic pain, including the impact of neuropathic lesions on RGS expression and, reciprocally, the influence of modifying RGS on GPCRs involved in the modulation of nociception as well as on the outcome of pain. In this context, we address the question of the relevance of RGS as promising targets in the treatment of neuropathic pain.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

A Precision Strategy to Cure Renal Cell Carcinoma by Targeting Transglutaminase 2.

In a recent report, no significance of transglutaminase 2 (TGase 2) was noted in the analyses of expression differences between normal and clear cell renal cell carcinoma (ccRCC), although we found that knock down of TGase 2 induced significant p53-mediated cell death in ccRCC. Generally, to find effective therapeutic targets, we need to identify targets that belong specifically to a cancer phenotype that can be differentiated from a normal phenotype. Here, we offer precise reasons why TGase 2 may be the first therapeutic target for ccRCC, according to several lines of evidence. TGase 2 is negatively regulated by von Hippel-Lindau tumor suppressor protein (pVHL) and positively regulated by hypoxia-inducible factor 1-α (HIF-1α in renal cell carcinoma (RCC). Therefore, most of ccRCC presents high level expression of TGase 2 because over 90% of ccRCC showed VHL inactivity through mutation and methylation. Cell death, angiogenesis and drug resistance were specifically regulated by TGase 2 through p53 depletion in ccRCC because over 90% of ccRCC express wild type p53, which is a cell death inducer as well as a HIF-1α suppressor. Although there have been no detailed studies of the physiological role of TGase 2 in multi-omics analyses of ccRCC, a life-long study of the physiological roles of TGase 2 led to the discovery of the first target as well as the first therapeutic treatment for ccRCC in the clinical field.

Hybridization of ß-Adrenergic Agonists and Antagonists Confers G Protein Bias.

Starting from the ß-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gαs, while they only show weak or even no ß-arrestin-2 recruitment at both ß1- and ß2-AR. Molecular dynamics simulations suggest that the difference in G protein efficacy and ß-arrestin recruitment of the hybrid ( S)-22, the full agonist epinephrine, and the ß2-selective, G protein-biased partial agonist salmeterol depends on specific hydrogen bonding between Ser5.46 and Asn6.55, and the aromatic head group of the ligands.

G proteins sculp root architecture in response to nitrogen in rice and Arabidopsis.

Nitrogen is a key nutrient for plant growth and development. Plants regulate nitrogen availability and uptake efficiency through controlling root architecture. While the heterotrimeric G protein complex is an important element to regulate root morphology, it remains unknown whether the G protein regulates the root architecture in response to nitrogen supply. We used rice and Arabidopsis G protein mutants to study the root architecture in response to different nitrogen concentrations. We found that nitrogen inhibits root horizontal projection area (network area), root perimeter, total length, but not root diameter (average root width). Nitrogen influenced bushiness and root spatial distribution by inhibiting horizontal growth and promoting vertical expansion. The dynamic changes of the rice G protein mutant DK22 at different concentrations of nitrogen from day 7 to day 9 were different from the wild type with regard to bushiness and spatial distribution. The agb1-2 mutant in Arabidopsis lacked the inhibitory effect of nitrate on root growth. The heterotrimeric G protein complex regulates the inhibitory effect on root growth caused by high nitrogen supply and root spatial distribution in response to different nitrogen concentrations.

Statins and bonehealth: a mini review / Estatinas y salud ósea: una minirevisión

Statins are a widely prescribed class of medications that inhibit similar pathways as the anti-resorptive bisphosphonate drugs. Statins target the mevalonate pathway by blocking HMG-CoA reductase. Several recent meta-analyses concluded statins are osteoprotective in the general population. Here we present current literature exploring the mechanisms underlying the putative osteoprotective effects of statins. We also review recent clinical studies, ranging from observational cohort studies to randomized clinical trials, testing the effect of statins on bone health in various populations. (AU)

Las estatinas son un grupo de drogas prescriptas en forma habitual, con la capacidad de bloquear vías de señalización similares a las inhibidas por los amino-bisfosfonatos. Las estatinas inhiben la vía del mevalonato, a través del bloqueo de diferentes enzimas. Varios metaanálisis recientes llevaron a la conclusión de que las estatinas tienen capacidad osteoprotectora en la población general. En esta revisión presentamos la literatura actual describiendo los mecanismos que subyacen en el potencial efecto osteoprotector de las estatinas, como así también estudios observacionales y clínicos aleatorizados sobre el efecto de estatinas en la salud ósea en diversas poblaciones. (AU)

Transglutaminase is a Critical Link Between Inflammation and Hypertension.

BACKGROUND: The pathogenesis of essential hypertension is multifactorial with different underlying mechanisms contributing to disease. We have recently shown that TNF superfamily member 14 LIGHT (an acronym for homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, also known as TNFSF14) induces hypertension when injected into mice. Research reported here was undertaken to examine the role of transglutaminase (TGase) in LIGHT-induced hypertension. METHODS AND RESULTS: Initial experiments showed that plasma and kidney TGase activity was induced by LIGHT infusion (13.91±2.92 versus 6.75±1.92 mU/mL and 19.86±3.55 versus 12.00±0.97 mU/10 µg) and was accompanied with hypertension (169±7.16 versus 117.17±11.57 mm Hg at day 14) and renal impairment (proteinuria, 61.33±23.21 versus 20.38±9.01 µg/mg; osmolality, 879.57±93.02 versus 1407.2±308.04 mmol/kg). The increase in renal TGase activity corresponded to an increase in RNA for the tissue TGase isoform, termed TG2. Pharmacologically, we showed that LIGHT-induced hypertension and renal impairment did not occur in the presence of cystamine, a well-known competitive inhibitor of TGase activity. Genetically, we showed that LIGHT-mediated induction of TGase, along with hypertension and renal impairment, was dependent on interleukin-6 and endothelial hypoxia inducible factor-1α. We also demonstrated that interleukin-6, endothelial hypoxia inducible factor-1α, and TGase are required for LIGHT-induced production of angiotensin receptor agonistic autoantibodies. CONCLUSIONS: Thus, LIGHT-induced hypertension, renal impairment, and production of angiotensin receptor agonistic autoantibodies require TGase, most likely the TG2 isoform. Our findings establish TGase as a critical link between inflammation, hypertension, and autoimmunity.

DHEA modulates the effect of cortisol on RACK1 expression via interference with the splicing of the glucocorticoid receptor.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). EXPERIMENTAL APPROACH: The THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific elisa. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRß. KEY RESULTS: DHEA induced a dose-related up-regulation of GRß and GRß knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. CONCLUSIONS AND IMPLICATIONS: These data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent.

ß-adrenoceptors as molecular targets in the treatment of hypertension.

Regulation of sympathoadrenal activity has been a long-time target in the management of hypertension. Regulation of ß-adrenoceptor (ßAR) function has been the most therapeutically important of these targets. The development of effective antihypertensive treatments based on ßAR antagonism paralleled the elucidation of the molecular basis of ß-adrenergic effects by the family of ßARs, which are members of the G-protein-coupled receptor (GPCR) superfamily. ßARs serve as the extracellular face of the transmembrane signalling pathway that results in the consequent activation of heterotrimeric G-proteins and the activation of several other newly appreciated signalling molecules that include ß-arrestins and GPCR kinases (GRKs). The aggregate effect of the activation of these signalling pathways mediates the response to ßAR activation. Paradoxically, the hypertensive state is characterized by impaired ßAR responsiveness. This defect is common to many other receptor systems linked to the stimulator G protein (Gs) and adenylyl cyclase activation. This impairment is principally mediated by receptor-G-protein uncoupling, which has been linked to increased expression and activity of GRK2.

Tissue transglutaminase in Alzheimer's disease: involvement in pathogenesis and its potential as a therapeutic target.

Protein misfolding and the formation of stable insoluble protein complexes by self-interacting proteins, in particular amyloid-ß and tau protein, play a central role in the pathogenesis of Alzheimer's disease (AD). Unfortunately, the underlying mechanisms that trigger the misfolding of self-interacting proteins that eventually results in formation of neurotoxic dimers, oligomers, and aggregates remain unclear. Elucidation of the driving forces of protein complex formation in AD is of crucial importance for the development of disease-modifying therapies. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that induces the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds, which results in both intra- and intermolecular protein cross-links. These tTG-catalyzed intermolecular cross-links induce stable, rigid, and insoluble protein complexes, whereas intramolecular cross-links change the conformation of proteins. Inhibition of tTG-catalyzed cross-linking counteracts the formation of protein aggregates, as observed in disease-models of other protein misfolding diseases, in particular Parkinson's and Huntington's diseases. Although data of tTG activity in AD models is limited, there is compelling evidence from both in vitro and postmortem human brain tissue of AD patients that point toward a crucial role for tTG in the pathogenesis of AD. Here, we review these data on the role of tTG in the initiation and development of protein aggregates in AD, and discuss the possibility to use inhibitors of the cross-linking activity of tTG as a new therapeutic approach for AD.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.

BACKGROUND: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. METHODS: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. RESULTS: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the µ and δ receptor subtypes revealed µ-receptor selectivity. CONCLUSION: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

Overexpression of developmentally regulated GTP-binding protein-2 increases bone loss.

The developmentally regulated GTP-binding protein-2 (DRG2) is a novel subclass of GTP-binding proteins. Many functional characteristics of osteoclasts (OC) are associated with small GTPases. We hypothesized that DRG2 affects bone mass via modulating OC activity. Using DRG2 transgenic mice, we investigated the role of DRG2 in bone remodeling. DRG2 overexpression caused a decrease in bone mass and an increase in the number and activity of OC in vivo. DRG2 overexpression increased fusion, spreading, survival, and resorption activity of OC in vitro. Downregulation of DRG2 by siRNA decreased fusion, spreading, and survival of OC, supporting the observations found in DRG2 transgenic OC. Transgenic mature OCs were larger, with actin rings and higher ERK, Akt, Rac1 and Rho activities than wild-type OCs. Inhibition of these proteins abolished the effects of DRG2 on formation of large OCs with actin rings, implying that DRG2 affects cytoskeleton reorganization in a Rac1/Rho/ERK/Akt-dependent manner. In summary, DRG2 is associated with survival and cytoskeleton organization of OC under influence of macrophage colony-stimulating factor, and its overexpression leads to elevated bone resorptive activity of OC, resulting in bone loss.

Effect of the G-protein ß3 subunit 825T allele on the change of body adiposity in obese female.

No clinical studies on the lipolytic effect of guanine nucleotide-binding protein ß3 subunit gene (GNB3) 825T polymorphism have been performed. This study was a subinvestigation of a 12-week randomized controlled trial (NCT01184560) for the additive effect of orlistat on sibutramine treatment. The analysis involved 101 obese females aged 18-49 years, genotyped at the GNB3 825 locus. To exclude any influence from potential confounders, we used an analysis of covariance model. After the intervention, fat mass proportion in total weight loss was significantly lower in subjects with a T allele than in those without a T allele (p = 0.034). GNB3 825T allele was associated with blunted fat mass reduction in obese females.

Peripubertal viral-like challenge and social isolation mediate overlapping but distinct effects on behaviour and brain interferon regulatory factor 7 expression in the adult Wistar rat.

A range of adverse, early life environmental influences such as viral infection and social deprivation are thought to increase risk of psychiatric illness later in life. Here, we used peripheral administration of the viral infection mimic polyriboinosinic-polyribocytidylic acid (polyI:C) to compare the consequences of peripubertal infection and isolation rearing. Isolation rearing induced deficits in sensorimotor gating and recognition memory while no changes in social interaction or spatial learning were observed. PolyI:C injection during the peripubertal period markedly increased expression of interferon-stimulated genes (Ifit2, Prkr, Mx2 and Irf7) in the hippocampal dentate gyrus demonstrating that peripheral administration of the viral mimic in the adolescent animal does have direct effects in the brain. Peripubertal infection mimicry induced a similar but later emerging behavioural deficit in prepulse inhibition implying the existence of a peripubertal window of opportunity for viral-mediated cytokine increases to impact brain development and function. PolyI:C treatment also impaired novel object recognition but did not alter spatial reference memory or social interaction. Combining the polyI:C challenge with social isolation did not exacerbate the behavioural deficits seen with isolation rearing alone. Using Irf7 as a marker, peripubertal viral infection mimicry, isolation rearing and a combination of both were all seen to produce a long-lasting molecular imprint on the interferon-associated signalling pathway in the principal neuron population of the hippocampal dentate gyrus. The data suggest that the sensitivity of brain structure and function to disruption by viral infection extends into the peripubertal period. Moreover, augmented interferon signalling in hippocampus may represent a common molecular imprint of environmental insults associated with neuropsychiatric illnesses like schizophrenia.

Role of pertussis toxin-sensitive G-protein, K+ channels, and voltage-gated Ca2+ channels in the antinociceptive effect of inosine.

Inosine is the first metabolite of adenosine. It exerts an antinociceptive effect by activating the adenosine A(1) and A(2A) receptors. We have previously demonstrated that inosine exhibits antinociceptive properties in acute and chronic mice models of nociception. The aim of this study was to investigate the involvement of pertussis toxin-sensitive G-protein-coupled receptors, as well as K(+) and Ca(2+) channels, in the antinociception promoted by inosine in the formalin test. Mice were pretreated with pertussis toxin (2.5 µg/site, i.t., an inactivator of G(i/0) protein); after 7 days, they received inosine (10 mg/kg, i.p.) or morphine (2.5 mg/kg, s.c., used as positive control) immediately before the formalin test. Another group of animals received tetraethylammonium (TEA) or 4-aminopyridine (4-AP) (1 µg/site, i.t., a non-specific voltage-gated K(+) channel blockers), apamin (50 ng/site, i.t., a small conductance Ca(2+)-activated K(+) channel blocker), charybdotoxin (250 pg/site, i.t., a large-conductance Ca(2+)-activated K(+) channel blocker), glibenclamide (100 µg/site, i.t., an ATP-sensitive K(+) channel blocker) or CaCl(2) (200 nmol/site, i.t.). Afterwards, the mice received inosine (10 mg/kg, i.p.), diclofenac (10 mg/kg, i.p., a positive control), or morphine (2.5 mg/kg, s.c., a positive control) immediately before the formalin test. The antinociceptive effect of inosine was reversed by the pre-administration of pertussis toxin (2.5 µg/site, i.t.), TEA, 4-aminopyridine, charybdotoxin, glibenclamide, and CaCl(2), but not apamin. Further, all K(+) channel blockers and CaCl(2) reversed the antinociception induced by diclofenac and morphine, respectively. Taken together, these data suggest that the antinociceptive effect of inosine is mediated, in part, by pertussis toxin-sensitive G-protein coupled receptors and the subsequent activation of voltage gated K(+) channel, large conductance Ca(2+)-activated and ATP-sensitive K(+) channels or inactivation of voltage-gated Ca(2+) channels. Finally, small conductance Ca(2+)-activated K(+) channels are not involved in the antinociceptive effect of inosine.

Inhibition of insulin secretion from rat pancreatic islets by dexmedetomidine and medetomidine, two sedatives frequently used in clinical settings.

The aim of this study was to determine whether dexmedetomidine (DEX) and medetomidine (MED), α2-adrenergic agonists clinically used as sedatives, influence insulin secretion from rat pancreatic islets. Islets were isolated from adult male Wistar rats after collagenase digestion. Static incubation was used to determine effects of DEX or MED on insulin secretion and ionic-channel currents of ß-cells. Results indicate that both drugs dose-dependently inhibit insulin secretion, DEX more potently than MED. The inhibitory effects were attenuated by addition of yohimbine or by pretreatment of rats with pertussis toxin (PTX). 10 nM DEX decreased the current amplitude of voltage-dependent Ca2+ channels, but this did not occur when the N-type Ca2+ channel blocker ω-conotoxin was added. In the presence of tetraethylammonium, a classical voltage-gated K+ channel (Kv channel) blocker, the magnitude of inhibition of insulin secretion by MED was reduced. However, when tolbutamide, a specific blocker of the ATP-sensitive K+ channel (KATP channel), was present, the magnitude of MED inhibition of insulin secretion was not influenced, suggesting that Kv-channel activity alteration, but not that of KATP channels, is involved in MED-associated insulin secretory inhibition. The Kv-channel currents were increased during 1 nM MED exposure at membrane potentials ranging from -30 mV to -10 mV, where action potentials were generated in response to glucose stimulation. These results indicate that DEX and MED inhibit insulin secretion through an α2-adrenoceptor and PTX-sensitive GTP-binding protein pathway that eventually involves Kv channel activation and Ca2+ channel inhibition.