Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats. Role of cannabinoid and vanilloid receptors

Background and aims: Anandamide is an endocannabinoid that evokes hypotension by interaction with peripheral cannabinoid CB1 receptors and with the perivascular transient receptor potential vanilloid type 1 protein (TRPV1). As anandamide has been implicated in the vasodilated state in advanced cirrhosis, the study investigated whether the mesenteric bed from cirrhotic rats has an altered and selective vasodilator response to anandamide. Methods: We assessed vascular sensitivity to anandamide, mRNA and protein expression of cannabinoid CB1 receptor and TRPV1 receptor, and the topographical distribution of cannabinoid CB1 receptors in resistance mesenteric arteries of cirrhotic and control rats. Results: Mesenteric vessels of cirrhotic animals displayed greater sensitivity to anandamide than control vessels. This vasodilator response was reverted by CB1 or TRPV1 receptor blockade, but not after endothelium denudation or nitric oxide inhibition. Anandamide had no effect on distal femoral arteries. CB1 and TRPV1 receptor protein was higher in cirrhotic than in control vessels. Neither CB1 mRNA nor protein was detected in femoral arteries. Immunochemistry showed that CB1 receptors were mainly in the adventitia and in the endothelial monolayer, with higher expression observed in vessels of cirrhotic rats than in controls. Conclusions: These results indicate that anandamide is a selective splanchnic vasodilator in cirrhosis which predominantly acts via interaction with two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in perivascular sensory nerve terminals of the mesenteric resistance arteries of these animals.

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4.

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle tocause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP14 were assayed on the activated mast cells. Betahexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogenactivated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by betahexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase,and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions: Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.

Motivational effects of cannabinoids are mediated by μ-opioid and k-opioid receptor

Repeated THC administration produces motivational and somaticadaptive changes leading to dependence in rodents. Toinvestigate the molecular basis for cannabinoid dependenceand its possible relationship with the endogenous opioid system,we explored Δ9-tetrahydrocannabinol (THC) activity in mice lacking μ-, δ- or κ-opioid receptor genes. Acute THCinduced hypothermia, antinociception, and ypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of μ receptors abolishes THC place preference. Deletion of κ receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of μ- and κ-opioid receptors in modulating reward pathways forms the basis for the dual euphoric–dysphoric activity of THC.

Synthesis, binding affinity and SAR of new benzolactam derivatives as dopamine D3 receptor ligands

A series of new benzolactam derivatives was synthesized and the derivatives were evaluated for theiraffinities at the dopamine D1, D2, and D3 receptors. Some of these compounds showed high D2 and/orD3 affinity and selectivity over the D1 receptor. The SAR study of these compounds revealed structuralcharacteristics that decisively influenced their D2 and D3 affinities. Structural models of the complexesbetween some of the most representative compounds of this series and the D2 and D3 receptors wereobtained with the aim of rationalizing the observed experimental results. Moreover, selected compoundsshowed moderate binding affinity on 5-HT2A which could contribute to reducing the occurrence of extrapyramidalside effects as potential antipsychotics.

Multi-receptor binding profile of clozapine and olanzapine: a structural study based on the new beta (2) adrenergic receptor template

Schizophrenia is a devastating mental disorder that has a largeimpact on the quality of life for those who are afflicted and isvery costly for families and society.[1] Although the etiology ofschizophrenia is still unknown and no cure has yet beenfound, it is treatable, and pharmacological therapy often producessatisfactory results. Among the various antipsychoticdrugs in use, clozapine is widely recognized as one ofthemost clinically effective agents, even if it elicits significant sideeffects such as metabolic disorders and agranulocytosis. Clozapineand the closely related compound olanzapine are goodexamples ofdrug s with a complex multi-receptor profile ;[2]they have affinities toward serotonin, dopamine, a adrenergic,muscarinic, and histamine receptors, among others.

Synthesis, binding affinity, and molecular docking analysis of new benzofuranone derivative as pontential antipsychotics

The complex etiology of schizophrenia has prompted researchers to develop clozapine-related multitargetstrategies to combat its symptoms. Here we describe a series of new 6-aminomethylbenzofuranones in aneffort to find new chemical structures with balanced affinities for 5-HT2 and dopamine receptors. Throughbiological and computational studies of 5-HT2A and D2 receptors, we identified the receptor serine residuesS3.36 and S5.46 as the molecular keys to explaining the differences in affinity and selectivity betweenthese new compounds for this group of receptors. Specifically, the ability of these compounds to establishone or two H-bonds with these key residues appears to explain their difference in affinity. In addition, wedescribe compound 2 (QF1004B) as a tool to elucidate the role of 5-HT2C receptors in mediating antipsychoticeffects and metabolic adverse events. The compound 16a (QF1018B) showed moderate to high affinitiesfor D2 and 5-HT2A receptors, and a 5-HT2A/D2 ratio was predictive of an atypical antipsychotic profile.

Nogo, myelin and axonal regeneration

Adult mammalian central nervous system (CNS) axons have very limited capacity of regrowth after injury. In recent years, advances in the field of axonal regeneration have proved that neurons do not regenerate, mainly because of the presence of inhibitory molecules. Myelin-associated proteins limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo, MAG and OMgp, share a common neuronal receptor (NgR), and together represent one of the main hindrances to neuronal regeneration. The recent molecular cloning of Nogo and its receptors opened a new door to the study of axon regeneration. However, many of the elements involved in the myelin inhibitory pathway are still unknown, and the preliminary experiments with knockout mice are rather contradictory. Because of this complexity, Nogo and NgR need to be characterized before precise strategies to promote axon regeneration in the CNS can be designed.

TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents intrkB (¿/¿) and trkC (¿/¿) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10¿P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21¿49%) and decreased densities of axonal varicosities (8¿17%) in both trkB (¿/¿) and trkC (¿/¿) mice. In addition, electron microscopic analyses showed thattrkB (¿/¿) and trkC (¿/¿) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (¿/¿) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

Constitutive expression of FGF4 disrupts the development of the eye and the anterior CNS during mouse embryogenesi, but does not influence the expressionof shh in these areas.

The biological consequences of constitutive fibroblast growth factor-4 (fgf4) expression have been analysed during anterior CNS development of mouse chimeric embryos. Severe mutant embryos exhibit exencephaly, absence of eye development and anomalous differentiation of neuropithelium. These embryos also show ectopic limb buds resembling the early phases of limb development. Because our results show that anterior CNS in those chimeric embrios does not express shh ectopically, we suggest that malformations may be due to interference between the ectopic expression of fgf4 in the cephalic area and the receptors for the members of the FGF family that regulate brain and eye development, namely fgf8. If this is correct, the results indirectly suport the crucial role of fgf8 in patterning the anterior CNS.

Nanomotors: new strategies of (bio)sensing based on motion

Nanomotors are nanoscale devices capable of converting energy into movement and forces. Among them, self-propelled nanomotors offer considerable promise for developing new and novel bioanalytical and biosensing strategies based on the direct isolation of target biomolecules or changes in their movement in the presence of target analytes. The mainachievements of this project consists on the development of receptor-functionalized nanomotors that offer direct and rapid target detection, isolation and transport from raw biological samples without preparatory and washing steps. For example, microtube engines functionalized with aptamer, antibody, lectin and enzymes receptors were used for the direct isolation of analytes of biomedical interest, including proteins and whole cells, among others. A target protein was also isolated from a complex sample by using an antigen-functionalized microengine navigating into the reservoirs of a lab-on-a-chip device. The new nanomotorbased target biomarkers detection strategy not only offers highly sensitive, rapid, simple and low cost alternative for the isolation and transport of target molecules, but also represents a new dimension of analytical information based on motion. The recognition events can be easily visualized by optical microscope (without any sophisticated analytical instrument) to reveal the target presence and concentration. The use of artificial nanomachines has shown not only to be useful for (bio)recognition and (bio)transport but also for detection of environmental contamination and remediation. In this context, micromotors modified with superhydrophobic layer demonstrated that effectively interacted, captured, transported and removed oil droplets from oil contaminated samples. Finally, a unique micromotor-based strategy for water-quality testing, that mimics live-fish water-quality testing, based on changes in the propulsion behavior of artificial biocatalytic microswimmers in the presence of aquatic pollutants was also developed. The attractive features of the new micromachine-based target isolation and signal transduction protocols developed in this project offer numerous potential applications in biomedical diagnostics, environmental monitoring, and forensic analysis.

Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways.

Smed454 dataset: unravelling the transcriptome of Schmidtea mediterranea

Background: Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. Results: In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. Conclusions: We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.

Smed454 dataset: unravelling the transcriptome of Schmidtea mediterranea

Background Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. Results In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. Conclusions We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.

Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal neuronal glial networks

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks

Developmental expression of the oligodendrocyte myelin glycoprotein in the mouse telencephalon

The oligodendrocyte myelin glycoprotein is a glycosylphosphatidylinositol-anchored protein expressed by neurons and oligodendrocytes in the CNS. Attempts have been made to identify the functions of the myelin-associated inhibitory proteins (MAIPs) after axonal lesion or in neurodegeneration. However, the developmental roles of some of these proteins and their receptors remain elusive. Recent studies indicate that NgR1 and the recently discovered receptor PirB restrict cortical synaptic plasticity. However, the putative factors that trigger these effects are unknown. Since Nogo-A is mostly associated with the endoplasmic reticulum and MAG appears late during development, the putative participation of OMgp should be considered. Here we examine the pattern of development of OMgp immunoreactive elements during mouse telencephalic development. OMgp immunoreactivity in the developing cortex follows the establishment of the thalamo-cortical barrel-field. At cellular level, we located OMgp neuronal membranes in dendrites and axons as well as in brain synaptosome fractions and axon varicosities. Lastly, the analysis of the barrel-field in OMgp-deficient mice revealed that although thalamo-cortical connections were formed, their targeting in layer IV was altered and numerous axons ectopically invaded layer II-III. Our data support the idea that early-expressed MAIPs play an active role during development and point to OMgp participating in thalamo-cortical connections.