Joan Sallés

Simultaneous determination of citalopram, fluoxetine and their main metabolites in human urine samples by solid-phase microextraction coupled with high-performance liquid chromatography

A liquid chromatography method was developed for the determination of some frequently prescribed selective serotonin re-uptake inhibitors (SSRI) - citalopram and fluoxetine - and its main metabolites - demethylcitalopram, didemethylcitalopram and norfluoxetine - in human urine samples, using a previous stage of solid-phase microextraction. All the extraction parameters influencing adsorption (extraction time, temperature, pH, ion strength and organic modifier addition) and desorption (desorption time and desorption solvent mixture composition) of the analytes on the fiber have been studied. A satisfactory reproducibility for extraction from urine samples (R.S.D.<10%) was obtained. The linearity for urine ranged from 0.05 to 2 mg l(-1) with limits of detection close to 0.01 mg l(-1), which cover the typical urinary concentrations obtained for citalopram, fluoxetine and their metabolites.

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain

G‐protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G‐protein‐coupled receptors such as the serotonin 5‐HT1A receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5‐HT1A receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co‐expression of GIRK subunits with the 5‐HT1A receptor and cell markers of glutamatergic, γ‐aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double‐label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double‐labeling experiments show that GIRK subunits are present in most of the 5‐HT1A receptor‐expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases. J. Comp. Neurol. 510:581–606, 2008.

Characterization of CB1 cannabinoid receptor immunoreactivity in postmortem human brain homogenates

The CB1 cannabinoid receptor (CB1) is the predominant type of cannabinoid receptor in the CNS, in which it displays a unique anatomical distribution and is present at higher densities than most other known seven transmembrane domain receptors. Nevertheless, as with almost all seven transmembrane domain receptors, the tertiary and quaternary structure of this receptor is still unknown. Studies of CB1 in rat cerebral tissue are scarce, and even less is known regarding the expression of CB1 in the human brain. Thus, the aim of the present work was to characterize CB1 expression in membranes from postmortem human brain using specific antisera raised against this protein. Western blot analysis of P1 and P2 fractions, and crude plasma membrane preparations from the prefrontal cortex showed that CB1 migrated as a 60 kDa monomer under reducing conditions. These data were confirmed by blotting experiments carried out with human U373MG astrocytoma cells as a positive control for CB1 expression and wild-type CHO cells as negative control. In addition, when proteins were solubilized in the absence of dithiothreitol, the anti-human CB1 antiserum detected a new band migrating at around 120 kDa corresponding in size to a putative CB1 dimer. This band was sensitive to reducing agents (50 mM dithiothreitol) and showed sodium dodecylsulphate stability, suggesting the existence of disulfide-linked CB1 dimers in the membrane preparations. Important differences in the anatomical distribution of CB1 were observed with regard to that described previously in monkey and rat; in the human brain, CB1 levels were higher in cortex and caudate than in the cerebellum.

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas.

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB(1) and CB(2) receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [(35)S]GTPgammaS binding. Western blot analysis showed that CB(1) receptor immunoreactivity was significantly lower in glioblastoma multiforme (-43%, n=10; p<0.05) than in normal post-mortem brain tissue (n=16). No significant differences were found for astrocytoma (n=6) and meningioma (n=8) samples. Conversely, CB(2) receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n=9; p<0.05) and astrocytoma (471%, n=4; p<0.05) than in control brain tissue (n=10). Finally, the maximal stimulation of [(35)S]GTPgammaS binding by WIN 55,212-2 was significantly lower in glioblastomas (134+/-4%) than in control membranes (183+/-2%; p<0.05). The basal [(35)S]GTPgammaS binding and the EC(50) values were not significantly different between both groups. The present results demonstrate opposite changes in CB(1) and CB(2) receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.

Reduced phospholipase C-β activity and isoform expression in the cerebellum of TS65DN mouse: A model of down syndrome

Agonist‐ and guanine‐nucleotide‐stimulated phospholipase C‐β (PLC) activity was characterized in crude plasma membrane preparations from cerebral cortex, hippocampus and cerebellum of Ts65Dn mice, a model for Down syndrome, and their control littermates. The levels of expression of PLC‐β(1–4) isoforms and G‐protein αq/11 subunits were also quantified by Western blot analysis to establish their contribution to the patterns of PLC functioning. PLC activity regulated by G‐proteins and muscarinic and 5‐HT2 receptors presented a regional distribution in both control and Ts65Dn mice. In both groups of mice, the intensity of PLC responses to maximal activation by calcium followed the sequence cerebellum > cortex > hippocampus. Both basal and maximal PLC activities, however, were significantly lower in cerebellar membranes of Ts65Dn than in control mice. This difference was mostly revealed in crude plasma membranes prepared from cerebellum at the level of G‐protein‐dependent‐PLC activity because the concentration‐response curve to GTPγS showed a reduction of the maximal effect in Ts65Dn mice, with no change in sensitivity (EC50). Western blot analysis showed a heterogeneous distribution of PLC‐β(1–4) isoforms in both groups of mice. The levels of PLC‐β4 isoform, however, were significantly lower in the cerebellum of Ts65Dn than in control mice. We conclude that the cerebellum of Ts65Dn mice has severe deficiencies in PLC activity stimulated by guanine nucleotides, which are specifically related to a lower level of expression of the PLC‐β4 isoform, a fact that may account for the neurological phenotype observed in this murine model of Down syndrome. J. Neurosci. Res. 66:540–550, 2001.

EBI2 regulates CXCL13-mediated responses by heterodimerization with CXCR5

B‐cell movement into lymphoid follicles depends on the expression of the chemokine receptor CXCR5 and the recently reported Epstein‐Barr virus‐induced receptor 2 (EBI2). In cooperation with CXCR5, EBI2 helps to position activated B cells in the follicle, although the mechanism is poorly understood. Using human HEK293T cells and fluorescence resonance energy transfer (FRET) techniques, we demonstrate that CXCR5 and EBI2 form homo‐ and heterodimers. EBI2 expression modulated CXCR5 homodimeric complexes, as indicated by the FRET50 value (CXCR5 homodimer, 0.9851±0.0784; CXCR5 homodimer+EBI2, 1.7320±0.4905; P<0.05). HEK293T cells expressing CXCR5/EBI2 and primary activated murine B cells both down‐modulated CXCR5‐mediated responses, such as Ca2+ flux, cell migration, and MAPK activation; this modulation did not occur when primary B cells were obtained from EBI2–/– mice. The mechanism involves a reduction in binding affinity of the ligand (CXCL13) for CXCR5 (KD: 5.05×10–8 M for CXCR5 alone vs. 1.49×10–7 M for CXCR5/EBI2) and in the efficacy (Emax) of G‐protein activation in CXCR5/EBI2‐coexpressing cells (42.33±4.3%; P<0.05). These findings identify CXCR5/EBI2 heterodimers as functional units that contribute to the plasticity of CXCL13‐mediated B‐cell responses.—Barroso, R., Muñoz, L. Martínez., Barrondo, S., Vega, B., Holgado, B. L., Lucas, P., Baíllo, A., Sallés, J., Rodríguez‐Frade J. M., Mellado, M. EBI2 regulates CXCL13‐mediated responses by heterodimerization with CXCR5. FASEB J. 26, 4841–4854 (2012). www.fasebj.org

Brain G protein-dependent signaling pathways in Down syndrome and Alzheimer's disease

Summary.Premature aging and neuropathological features of Alzheimer’s disease (AD) are commonly observed in Down syndrome (DS). Based on previous findings in a DS mouse model, the function of signaling pathways associated with adenylyl cyclase (AC) and phospholipase C (PLC) was assessed in cerebral cortex and cerebellum of age-matched adults with DS, AD, and controls. Basal production of cAMP was reduced in DS but not in AD cortex, and in both, DS and AD cerebellum. Responses to GTPγS, noradrenaline, SKF 38393 and forskolin were more depressed in DS than in AD cortex and cerebellum. Although no differences in PLC activity among control, DS and AD cortex were observed under basal and GTPγS- or Ca-stimulated conditions, the response of DS cortex to serotonergic and cholinergic stimulation was depressed, and that of AD was only impaired at cholinergic stimulation. No differences were documented in cerebellum. Our results demonstrate that PLC and AC were severely disturbed in the aged DS and AD brains, but the alterations in DS were more severe, and differed to some extent from those observed in AD.

Regulation of phospholipase Cβ activity by muscarinic acetylcholine and 5-HT2 receptors in crude and synaptosomal membranes from human cerebral cortex

Abstract Stimulation of phospholipase Cβ by receptor agonists and G proteins has been characterized in crude cerebral membrane preparations, but little is known about their presynaptic localizations and little information is currently available for human brain tissue. The characteristics of phosphoplipase C transmembrane signaling were studied in crude and synaptosomal plasma membranes from postmortem human prefrontal cortex by measuring the hydrolysis of exogenous [ 3 H]phosphatidylinositol4,5bisphosphate(PIP 2 ) and the immunoreactive levels of phospholipase C (PLC) and G αq/11 proteins. Regulation of PLC activity by Ca 2+ and the 5-HT 2 receptor agonist 5-methyltryptamine, but not by guanosine 5′- O -[3-thiotriphosphate] and the muscarinic acetylcholine receptor agonist carbachol were different between crude and synaptosomal membranes. KCl (20 mM) stimulation was absent in both preparations. Levels of G αq/11 -protein subunits differed between preparations. The functional inhibition carried out with pirenzepine in crude membranes in order to reverse the carbachol-induced PLC stimulation indicates the existence of a component (53%) of the response that is activated by the M 1 muscarinic acetylcholine receptor subtype, and another component (47%) probably mediated by the M 3 muscarinic acetylcholine receptor subtype. In synaptosomal plasma membranes an increased inhibition of carbachol-induced PLC activation through M 1 was found. The PLC activation by 5-methyltryptamine (ketanserin-sensitive in crude membranes) was absent in synaptosomal plasma membranes suggesting the lack of activity mediated by 5-HT 2 -serotonin receptors.

Nuclear phospholipase C-β1 and diacylglycerol LIPASE-α in brain cortical neurons

Phosphoinositide (PtdIns) signaling involves the generation of lipid second messengers in response to stimuli in a receptor-mediated manner at the plasma membrane. In neuronal cells of adult brain, the standard model proposes that activation of metabotropic receptors coupled to Phospholipase C-β1 (PLC-β1) is linked to endocannabinoid signaling through the production of diacylglycerol (DAG), which could be systematically metabolized by 1,2-diacylglycerol Lipases (DAGL) to produce an increase of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. However, the existence of a nuclear PtdIns metabolism independent from that occurring elsewhere in the cell is now widely accepted, suggesting that the nucleus constitutes both a functional and a distinct compartment for PtdIns metabolism. In this review, we shall highlight the main achievements in the field of neuronal nuclear inositol lipid metabolism with particular attention to progress made linked to the 2-AG biosynthesis. Our aim has been to identify potential sites of 2-AG synthesis other than the neuronal cytoplasmic compartment by determining the subcellular localization of PLC-β1 and DAGL-α, which is much more abundant than DAGL-β in brain. Our data show that PLC-β1 and DAGL-α are detected in discrete brain regions, with a marked predominance of pyramidal morphologies of positive cortical cells, consistent with their role in the biosynthesis and release of 2-AG by pyramidal neurons to control their synaptic inputs. However, as novelty, we showed here an integrated description of the localization of PLC-β1 and DAGL-α in the neuronal nuclear compartment. We discuss our comparative analysis of the expression patterns of PLC-β1 and DAGL-α, providing some insight into the potential autocrine role of 2-AG production in the neuronal nuclear compartment that probably subserve additional roles to the recognized activation of the CB1 cannabinoid receptor.

Cellular neurochemical characterization and subcellular localization of phospholipase C β1 in rat brain

The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCβ1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCβ1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCβ1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCβ1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCβ1 was similar (around 2/3) in layers I, II-III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCβ1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCβ1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCβ1 involved specifically one of the two splice variants of PLCβ1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCβ1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCβ1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.