Unraveling monoamine receptors involved in the action of typical and atypical antipsychotics on glutamatergic and serotonergic transmission in prefrontal cortex

El pdf del artículo es la versión post-print.The systemic administration of noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists has been considered as a pharmacological model of schizophrenia. In the present work, we used in vivo microdialysis to examine: first, the effects of MK-801, on the efflux of glutamate and serotonin (5-HT) in the medial prefrontal cortex (mPFC) of the rat; second, whether the MK-801-induced changes in the cortical efflux of both transmitters could be blocked by atypical (clozapine and olanzapine) and classical (haloperidol and chlorpromazine) antipsychotic drugs given intra-mPFC; and third, the role of local blockade of dopamine D2/D3/D4, serotonin 5-HT2A and α1-adrenergic receptors as well as agonism at dopamine D1/D5 and 5-HT1A receptors in the mPFC on the increased efflux of glutamate and 5-HT elicited by MK-801. The four antipsychotic drugs blocked the MK-801-induced increase in glutamate, whereas only clozapine and olanzapine were able to block the increased efflux of 5-HT. Furthermore, M100907 (5-HT2A antagonist), BAY x 3702 (5-HT1A agonist) and prazosin (α1-adrenergic antagonist) blocked the MK-801-induced increase of 5-HT and glutamate in the mPFC. In contrast, raclopride (D2/D3 antagonist) and L-745,870 (D4 antagonist) were able to prevent the increased efflux of glutamate (but not that of 5-HT) elicited by MK-801. SKF-38393 (dopamine D1/D5 agonist) also prevented the MK-801-induced increase of glutamate in the mPFC, but the same effect on cortical 5-HT was reached only at the highest concentration tested. We suggest that the blockade of an exacerbated 5-HT release in the mPFC induced by NMDA antagonists can be a characteristic of atypical antipsychotic drugs. Moreover, we propose that D 2/D3/D4 receptor antagonists would act predominantly on a subpopulation of GABAergic interneurons of the mPFC, thus enhancing cortical inhibition, which would prevent an excessive glutamatergic transmission. Dopamine D1/D5 agonists would further stimulate GABA release from other subpopulation of interneurons controlling cortical output to the dorsal raphe nucleus. Atypical antipsychotic drugs might further act upon 5-HT2A, 5-HT1A and α1- adrenoceptors present in pyramidal cells (including those projecting to the dorsal raphe nucleus), which would directly inhibit an excessive excitability of these cells. © 2010 Bentham Science Publishers Ltd.This work was supported by the Spanish Ministry of Health (FIS Grant PI070111 to A. A.), the Spanish Ministry of Education and Science (Grant SAF 2007-62378 to F.A.), and the Generalitat de Catalunya (SGR2005/00758). X.L.- G. is the recipient of a predoctoral fellowship from the Consejo Superior de Investigaciones Cientificas (CSIC).Peer Reviewe

Structural and functional analysis of tomato sterol C22 desaturase

Background: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function. Results: In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding. Conclusions: Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism

Clozapine and haloperidol differently suppress the MK-801-increased glutamatergic and serotonergic transmission in the medial prefrontal cortex of the rat

The administration of noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine and ketamine has been shown to increase the extracellular concentration of glutamate and serotonin (5-HT) in the medial prefrontal cortex (mPFC). In the present work, we used in vivo microdialysis to examine the effects of the more potent noncompetitive NMDA receptor antagonist, MK-801, on the efflux of glutamate and 5-HT in the mPFC, and whether the MK-801-induced changes in the cortical efflux of both transmitters could be blocked by clozapine and haloperidol given systemically or intra-mPFC. The systemic, but not the local administration of MK-801, induced an increased efflux of 5-HT and glutamate, which suggests that the NMDA receptors responsible for these effects are located outside the mPFC, possibly in GABAergic neurons that tonically inhibit glutamatergic inputs to the mPFC. The MK-801-induced increases of extracellular glutamate and 5-HT were dependent on nerve impulse and the activation of mPFC AMPA/kainate receptors as they were blocked by tetrodotoxin and NBQX, respectively. Clozapine and haloperidol blocked the MK-801-induced increase in glutamate, whereas only clozapine was able to block the increased efflux of 5-HT. The local effects of clozapine and haloperidol paralleled those observed after systemic administration, which emphasizes the relevance of the mPFC as a site of action of these antipsychotic drugs in offsetting the neurochemical effects of MK-801. The ability of clozapine to block excessive cortical 5-HT efflux elicited by MK-801 might be related to the superior efficacy of this drug in treating negative/cognitive symptoms of schizophrenia.This work was supported by the Spanish Ministry of Education and Science Grants SAF 2004-05525 and SAF 2003-04930 and by the Generalitat de Catalunya (SGR2005/00758 and SGR2005/00826). XL-G, ZB, and MA-B were recipients of predoctoral fellowships from the Consejo Superior de Investigaciones Científicas (CSIC), Spanish Ministry of Education and Science, and Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), respectively.Peer reviewe

Complex interplays between phytosterols and plastid development

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C₅ precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism

Culinary and sensory traits diversity in the Spanish Core Collection of common beans (Phaseolus vulgaris L.)

The Spanish National Plant Genetic Resource Center's core collection of bean germplasm includes 202 accessions selected from more than 3000 accessions in function of passport data, seed phenotype, genetic background, and agronomic traits. To acquire more useful information about these accessions, we cultivated and characterized them for sensory and culinary traits. We found considerable variation for culinary and sensory traits of the cooked beans (mean coefficients of variation: 41% for the sensory traits and 40% for the culinary traits). The large dataset enabled us to study correlations between sensory and culinary traits and among these traits and geographic origin, seed color, and growth habit. Greater proportion of white in the seed coat correlated positively with brightness and negatively with mealiness (r=0.60, r=-0.60, p<0.001, respectively). Mealiness correlated negatively with seed-coat roughness and rate of water absorption (r=-0.60, r=-0.53, p<0.001, respectively). Materials of Andean origin had lower seed-coat brightness (p<0.01) and seed-coat roughness, and greater seed-coat perceptibility, mealiness, flavor, and aroma (p<0.001) than materials of Mesoamerican origin. Growth habit failed to correlate with culinary or sensory traits.Postprint (published version

Inactivation of UDP-Glucose Sterol Glucosyltransferases Enhances Arabidopsis Resistance to Botrytis cinerea

Altres ajuts: Universitat Jaume I (UJI-B2016-43)Free and glycosylated sterols are both structural components of the plasma membrane that regulate their biophysical properties and consequently different plasma membrane-associated processes such as plant adaptation to stress or signaling. Several reports relate changes in glycosylated sterols levels with the plant response to abiotic stress, but the information about the role of these compounds in the response to biotic stress is scarce. In this work, we have studied the response to the necrotrophic fungus Botrytis cinerea in an Arabidopsis mutant that is severely impaired in steryl glycosides biosynthesis due to the inactivation of the two sterol glucosyltransferases (UGT80A2 and UGT80B1) reported in this plant. This mutant exhibits enhanced resistance against B. cinerea when compared to wild-type plants, which correlates with increased levels of jasmonic acid (JA) and up-regulation of two marker genes (PDF1.2 and PR4) of the ERF branch of the JA signaling pathway. Upon B. cinerea infection, the ugt80A2;B1 double mutant also accumulates higher levels of camalexin, the major Arabidopsis phytoalexin, than wild-type plants. Camalexin accumulation correlates with enhanced transcript levels of several cytochrome P450 camalexin biosynthetic genes, as well as of their transcriptional regulators WRKY33, ANAC042, and MYB51, suggesting that the Botrytis-induced accumulation of camalexin is coordinately regulated at the transcriptional level. After fungus infection, the expression of genes involved in the indole glucosinolate biosynthesis is also up-regulated at a higher degree in the ugt80A2;B1 mutant than in wild-type plants. Altogether, the results of this study show that glycosylated sterols play an important role in the regulation of Arabidopsis response to B. cinerea infection and suggest that this occurs through signaling pathways involving the canonical stress-hormone JA and the tryptophan-derived secondary metabolites camalexin and possibly also indole glucosinolates

Tomato UDP-glucose sterol glycosyltransferases: A family of developmental and stress regulated genes that encode cytosolic and membrane-associated forms of the enzyme

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate

3DHIP-calculator-A new tool to stochastically assess deep geothermal potential using the heat-in-place method from voxel-based 3D geological models

The assessment of the deep geothermal potential is an essential task during the early phases of any geothermal project. The well-known "Heat-In-Place" volumetric method is the most widely used technique to estimate the available stored heat and the recoverable heat fraction of deep geothermal reservoirs at the regional scale. Different commercial and open-source software packages have been used to date to estimate these parameters. However, these tools are either not freely available, can only consider the entire reservoir volume or a specific part as a single-voxel model, or are restricted to certain geographical areas. The 3DHIP-Calculator tool presented in this contribution is an open-source software designed for the assessment of the deep geothermal potential at the regional scale using the volumetric method based on a stochastic approach. The tool estimates the Heat-In-Place and recoverable thermal energy using 3D geological and 3D thermal voxel models as input data. The 3DHIP-Calculator includes an easy-to-use graphical user interface (GUI) for visualizing and exporting the results to files for further postprocessing, including GIS-based map generation. The use and functionalities of the 3DHIP-Calculator are demonstrated through a case study of the Reus-Valls sedimentary basin (NE, Spain)