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Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT(2B), is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT(2B) receptors by circulating c-kit(+) precursor cells, whereas mice lacking 5-HT(2B) receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT(2B) receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT(2B) receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34(+) or mice c-kit(+) progenitor cells in the presence of a 5-HT(2B) receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT(2B) receptors on bone marrow lineage progenitors is critical for the development of PAH.

Peripheral serotonin, synthesized by tryptophan hydroxylase-1 (TPH(1)), has been shown to play a key role in several physiological functions. Recently, controversy has emerged about whether peripheral serotonin has any effect on bone density and remodeling.We therefore decided to investigate in detail bone remodeling in growing and mature TPH(1) knockout mice (TPH(1)(-/-)). Bone resorption in TPH(1)(-/-) mice, as assessed by biochemical markers and bone histomorphometry, was markedly decreased at both ages. Using bone marrow transplantation, we present evidence that the decrease in bone resorption in TPH(1)(-/-) mice is cell-autonomous. Cultures from TPH(1)(-/-) in the presence of macrophage colony-stimulating factor and receptor activator for NF-KB ligand (RANKL) displayed fewer osteoclasts, and the decreased differentiation could be rescued by adding serotonin. Our data also provide evidence that in the presence of RANKL, osteoclast precursors express TPH(1) and synthesize serotonin. Furthermore, pharmacological inhibition of serotonin receptor 1B with SB224289, and of receptor 2A with ketanserin, also reduced the number of osteoclasts. Our findings reveal that serotonin has an important local action in bone, as it can amplify the effect of RANKL on osteoclastogenesis.

Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of β1 integrins at the plasma membrane. β1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.

The aim of the present study was to examine the effect of sympathectomy on plasmatic and arterial native and oxLDL levels, as well as arterial LDL receptors (LDLR) and scavenger receptors in hypercholesterolemic rats, which are normally protected against atherosclerosis. Neonatal Wistar rats received subcutaneous injections of either guanethidine for sympathectomy (Gua+HC) or vehicle (HC), then were fed 1% cholesterol for three months. Intact normocholesterolemic rats were used as control of the HC group. Total cholesterol (TC) and LDL-cholesterol were evaluated in the plasma and the abdominal aorta by an auto-analyzer. Plasmatic and aortic oxLDL and native LDL-apo B100 were assessed by a sandwich ELISA. Aortic and hepatic native LDLR and aortic scavenger receptors (CD36 and SR-A) were quantified at mRNA and protein levels by real time PCR and western immunoblot. The effect of hypercholesterolemia was limited to an increase in plasmatic TC and LDL-cholesterol and a decrease in aortic apoB100 and aortic and hepatic LDLR. Hypercholesterolemia and sympathectomy in combination increased markedly plasmatic and aortic TC, LDL-cholesterol, apo B100 and oxLDL together with aortic scavenger receptors, but reduced markedly aortic and hepatic LDLR. Sympathectomy broke down the rat's protection against hypercholesterolemia by promoting accumulation of native and oxLDL in the aorta via scavenger receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Supporting the hypothesis that proteasome dysfunction is involved in Parkinson's disease (PD), McNaught et al. (2004) reported that the systemic administration of the proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leu-aldehyde (PSI) in rats led to the degeneration of the nigrostriatal pathway. However, several groups could not reproduce this finding. We herein attempted to improve the reliability of the PSI model by chronically delivering the inhibitor using osmotic minipumps in aged mice. We also tested whether PSI co-administered with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) could act synergistically to induce toxicity. We found that PSI produced a significant reduction in locomotor activity that was mildly exacerbated by MPTP. However, PSI alone produced no sign of degeneration of the nigrostriatal dopaminergic pathway and did not exacerbate MPTP toxicity. To conclude, PSI administration does not provide a reliable phenotypic model of PD., (Copyright © 2009 Elsevier Inc. All rights reserved.)

We previously showed that sympathectomy induces thickened intima and decreases the expression of cytoskeletal proteins associated with a differentiated smooth muscle cell (SMC) phenotype in hypercholesterolemic rats. In the present study, we sought to determine the effect of sympathectomy on various components of the extracellular matrix (ECM) in the aorta from these animals, since the state of SMC differentiation depends on the nature of ECM components. Collagen types I and III, previously reported to be associated with SMC dedifferentiation, and collagen VI, elastin, laminin and elastin-laminin receptor (E/L-R), previously reported to be associated with SMC differentiation, were analyzed by western immunoblot and confocal microscopy in abdominal aortae from sham rats and hypercholesterolemic rats sympathectomized with guanethidine. Both western immunoblot and immunohistological analysis showed an increase in collagens I and III (more for collagen I), with abundant labeling in the media, adventitia and thickened intima in sympathectomized aortae. Collagen IV labeling was decreased in the media and adventitia and was weak in the thickened intima in sympathectomised aortae. The E/L-R increased and was abundantly labeled in the media and weakly in the thickened intima in sympathectomized aortae. Elastin and laminin decreased and appeared less labeled in the media in the sympathectomised aortae. In the thickened intima, laminin was slightly labeled while elastin was not obviously labeled. These data show that sympathectomy favors the ECM features reported in association with a dedifferentiated/immature SMC phenotype and intimal thickening, probably by actions on both SMCs and fibroblasts., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Behavioral syndromes are suites of two or more behaviors that correlate across environmental contexts. The aggression-boldness syndrome links aggression, boldness, and exploratory activity in a novel environment. Although aggression-boldness has been described in many animals, the mechanism linking its behavioral components is not known. Here we show that mutation of the gene encoding fibroblast growth factor receptor 1a (fgfr1a) simultaneously increases aggression, boldness, and exploration in adult zebrafish. We demonstrate that altered Fgf signaling also results in reduced brain histamine levels in mutants. Pharmacological increase of histamine signaling is sufficient to rescue the behavioral phenotype of fgfr1a mutants. Together, we show that a single genetic locus can underlie the aggression-boldness behavioral syndrome. We also identify one of the neurotransmitter pathways that may mediate clustering of these behaviors.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

We previously reported that confronting mice to the Unpredictable Chronic Mild Stress procedure (UCMS) resulted in peripheral and cerebral alterations of the kynurenine pathway (KP). The present study tested whether KP disturbances are associated with differences in anxiety- and depressive-like behaviors in both naïve and UCMS mice. Non-stressed and UCMS mice were subjected to the elevated plus maze test and to the forced swim test. Mice were then sacrificed for quantification of tryptophan (TRP)-serotonin (5-HT) and TRP-kynurenine (KYN) metabolites in two corticolimbic structures involved in the regulation of mood (cingulate cortex=CC; amygdala=AMY). We showed that an elevated peripheral (lung) KYN/TRP ratio is correlated to the magnitude of anxiodepressive-like phenotypes only in UCMS mice. We also observed, in UCMS mice, that a high peripheral (lung) KYN/TRP ratio is associated with an increased metabolism of 5-HT in CC and a reduced level of kynurenic acid (KYNA) in AMY. Our results suggest that elevated peripheral KP might underlie cerebral biochemical changes and might consequently be involved in the modulation of behavior but only in UCMS mice. These findings make more complete the comprehension of the KP involvement in behavioral changes induced by chronic stress and suggest that it could play a crucial role in the modulation of emotional states., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The now-banned anorectic molecule, dexfenfluramine, promotes serotonin release through a serotonin transporter-dependent mechanism, and it has been widely prescribed for the treatment of obesity. Previous studies have identified that 5-HT(2B) receptors have important roles in dexfenfluramine side effects, that is, pulmonary hypertension, plasma serotonin level regulation, and valvulopathy. We thus investigated a putative contribution of 5-HT(2B) receptors in dexfenfluramine-dependent feeding behavior in mice. Interestingly, the hypophagic response to dexfenfluramine (3-10 mg/kg) observed in wild-type mice (1-4 h) was eliminated in mice lacking 5-HT(2B) receptors (5-HT(2B)(-/-)). These findings were further validated by the lack of hypophagic response to dexfenfluramine in wild-type mice treated with RS127445, a highly selective and potent antagonist (pKi=8.22 ± 0.24). Using microdialysis, we observed that in 5-HT(2B)(-/-) awake mice, the dexfenfluramine-induced hypothalamic peak of serotonin release (1 h) was strongly reduced (fourfold) compared with wild type. Moreover, using hypothalamic synaptosomes, we established the serotonergic neuron autonomous properties of this effect: a strong serotonin release was observed upon dexfenfluramine stimulation of synaptosome preparation from wild type but not from mice lacking active 5-HT(2B) receptors. These findings strongly suggest that activation of presynaptic 5-HT(2B) receptors is a limiting step in the serotonin transporter dependent-releasing effect of dexfenfluramine, whereas other serotonin receptors act downstream with respect to feeding behavior.

Occurring both peripherally and centrally, the kynurenine pathway (KP) - an alternative pathway to 5-HT synthesis from tryptophan (TRP) - could be of particular value to better understand the link between peripheral changes of circulating levels of glucocorticoids (GC)/proinflammatory cytokines and altered neurotransmission observed in depressed patients. Indeed, it is activated by these mediators of stress and can produce several neuroactive compounds like quinolinic acid (QUIN) and kynurenic acid (KYNA) that can respectively increase and decrease glutamate concentration in brain. In order to characterize the role of both the peripheral and cerebral KP in the pathophysiology of depressive disorders, we used the Unpredictable Chronic Mild Stress (UCMS) to induce a depressive-like syndrome and we then measured the level of relevant TRP-KYN pathway metabolites: KYN, 3-hydroxykynurenine (3HK; precursor of QUIN) and KYNA. We also measured TRP-5HT pathway metabolites: TRP, 5-HT, 5-HIAA. We showed that UCMS increased TRP catabolism along the KP in the periphery. 5-HT and KYN were found to be strongly negatively correlated in all brain structures of control mice and of UCMS mice except in the hippocampus. More importantly we found that KYN was preferentially metabolized along the QUIN pathway at the subcortical level (amygdala/striatum) whereas, at the cortical level (cingulate cortex), the QUIN pathway was reduced. Considering the role of these metabolites on the glutamatergic neurotransmission, we propose that such KP alterations could participate to the cortical/subcortical glutamatergic alterations reported in depressed patients.

The effect of sympathectomy and sensory denervation on vascular smooth muscle cell (SMC) differentiation was investigated in hypercholesterolemic rats. Newborn rats received injections of guanethidine, capsaicin or both for denervations. Shams received injections of vehicles. The four groups were fed 1% cholesterol diet for 3 months. Intact normocholesterolemic rats were also exploited. Serum total cholesterol and systolic blood pressure (SBP) were measured. Lipid presence in the arterial wall was shown by Red-Oil-O staining. Catecholamine- and CGRP-containing fibres, vimentin and the adult SMC markers alpha-SMC-actin, desmin and h-caldesmon were analysed in abdominal aorta by western blot and confocal microscope. The sympathetic (catecholamine) fibres and SBP increased after sensory denervation while the sensory (CGRP) fibres increased and SBP decreased after sympathectomy. SBP was not changed after double denervation. Total cholesterol increased in sham and rose further after sympathectomy. Vimentin and the three adult SMC markers were not influenced by hypercholesterolemia. However, in the sympathectomized aorta, vimentin increased, desmin did not change, whereas alpha-SMC-actin and h-caldesmon decreased. In the sensory-denervated aorta, vimentin decreased, desmin increased, alpha-SMC-actin did not change and h-caldesmon decreased but less than in sympathectomized aorta. In the doubly denervated aorta, vimentin did not change and the three adult SMC markers decreased, although less than in sympathectomized aorta for alpha-SMC-actin and h-caldesmon. Thickened intima was identified by Red-Oil-O staining in the sympathectomized and (less remarkably) doubly denervated aortas containing SMCs not fully dedifferentiated. Our findings suggest that sympathectomy induces intimal thickening and favours SMC dedifferentiation, whereas sensory denervation favours SMC differentiation.

Neuropathological studies have reported a strong neurofibrillary degeneration of the tuberomamillary nucleus, the region of origin of histamine neurons, in Alzheimer's disease (AD). Histaminergic neurons enhance cognition and memory, suggesting that their degeneration may contribute to the cognitive decline of AD. Besides neurons, the brain histaminergic system comprises mast cells and microglia that can also produce histamine. The level of activity of this histaminergic system in AD remained unknown. In the present study, we have measured the levels of the main histamine metabolite in brain, tele-methylhistamine (t-MeHA), an index of histaminergic system activity, in the cerebrospinal fluid (CSF) of 97 non-AD (controls) and 91 AD patients, males or females. t-MeHA levels in CSF of controls tended to be higher, although non-significantly, in females than in males. t-MeHA levels of controls and AD significantly increased with age (1.66 ± 0.13, 2.04 ± 0.12, and 2.76 ± 0.12 pmol/ml at 40, 60 and 80 years, respectively). In spite of the strong degeneration of histamine neurons in the disease, t-MeHA levels in CSF were only slightly decreased in AD compared to controls (2.14 ± 0.10 vs 2.76 ± 0.13 pmol/ml, -22%, p < 0.01). This decrease was similar whatever the age, and was slightly higher in females than in males. The increase observed with age, and the limited magnitude of the decrease in AD even at late stages may result from the compensatory activation of spared neurons, as well as the neuroinflammation-induced activation of microglia occurring in senescence and AD.

Background: Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years., Methodology/principal Findings: 5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)--the 5-HT/MAO catabolite--in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity., Conclusions/significance: This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health.

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) reverses dopamine and serotonin transporters to produce efflux of dopamine and serotonin, respectively, in regions of the brain that have been implicated in reward. However, the role of serotonin/dopamine interactions in the behavioral effects of MDMA remains unclear. We previously showed that MDMA-induced locomotion, serotonin and dopamine release are 5-HT(2B) receptor-dependent. The aim of the present study was to determine the contribution of serotonin and 5-HT(2B) receptors to the reinforcing properties of MDMA.We show here that 5-HT(2B) (-/-) mice do not exhibit behavioral sensitization or conditioned place preference following MDMA (10 mg/kg) injections. In addition, MDMA-induced reinstatement of conditioned place preference after extinction and locomotor sensitization development are each abolished by a 5-HT(2B) receptor antagonist (RS127445) in wild type mice. Accordingly, MDMA-induced dopamine D1 receptor-dependent phosphorylation of extracellular regulated kinase in nucleus accumbens is abolished in mice lacking functional 5-HT(2B) receptors. Nevertheless, high doses (30 mg/kg) of MDMA induce dopamine-dependent but serotonin and 5-HT(2B) receptor-independent behavioral effects.These results underpin the importance of 5-HT(2B) receptors in the reinforcing properties of MDMA and illustrate the importance of dose-dependent effects of MDMA on serotonin/dopamine interactions.

By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT(2B) receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT(2B) receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT(2B) receptor solely in cardiomyocytes, like global 5-HT(2B) receptor-null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT(1)Rs and 5-HT(2B) receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II-dependent cytokine production occur via a Src/heparin-binding epidermal growth factor-dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT(2B) receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT(1) and 5-HT(2B) receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT(2B) receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8+ and CD4+ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1-/- and Tcrb-/- mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1-/- mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1-/- mice reconstituted with IFN-gamma-deficient splenocytes were not protected. These data indicate that T cell-mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells and requires the expression of FasL but not IFNgamma. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.

Organic cation transporters (OCTs) are carrier-type polyspecific permeases known to participate in low-affinity extraneuronal catecholamine uptake in peripheral tissues. OCT3 is the OCT subtype most represented in the brain, yet its implication in central aminergic neurotransmission in vivo had not been directly demonstrated. In a detailed immunohistochemistry study, we show that OCT3 is expressed in aminergic pathways in the mouse brain, particularly in dopaminergic neurons of the substantia nigra compacta, non-aminergic neurons of the ventral tegmental area, substantia nigra reticulata (SNr), locus coeruleus, hippocampus and cortex. Although OCT3 was found mainly in neurons, it was also occasionally detected in astrocytes in the SNr, hippocampus and several hypothalamic nuclei. In agreement with this distribution, OCT3/Slc22a3-deficient mice show evidence of altered monoamine neurotransmission in the brain, with decreased intracellular content and increased turnover of aminergic transmitters. The behavioral characterization of these mutants reveal subtle behavioral alterations such as increased sensitivity to psychostimulants and increased levels of anxiety and stress. Altogether our data support a role of OCT3 in the homeostatic regulation of aminergic neurotransmission in the brain.

The "club drug" 3,4-methylenedioxymethamphetamine (MDMA; also known as ecstasy) binds preferentially to and reverses the activity of the serotonin transporter, causing release of serotonin [5-hydroxytryptamine (5-HT)] stores from nerve terminals. Subsequent activation of postsynaptic 5-HT receptors by released 5-HT has been shown to be critical for the unique psychostimulatory effects of MDMA. In contrast, the effects of direct activation of presynaptic and/or postsynaptic receptors by MDMA have received far less attention, despite the agonist actions of the drug itself at 5-HT(2) receptors, in particular the 5-HT(2B) receptor. Here we show that acute pharmacological inhibition or genetic ablation of the 5-HT(2B) receptor in mice completely abolishes MDMA-induced hyperlocomotion and 5-HT release in nucleus accumbens and ventral tegmental area. Furthermore, the 5-HT(2B) receptor dependence of MDMA-stimulated release of endogenous 5-HT from superfused midbrain synaptosomes suggests that 5-HT(2B) receptors act, unlike any other 5-HT receptor, presynaptically to affect MDMA-stimulated 5-HT release. Thus, our findings reveal a novel regulatory component in the actions of MDMA and represent the first demonstration that 5-HT(2B) receptors play an important role in the brain, i.e., modulation of 5-HT release. As such, 5-HT(2B) receptor antagonists may serve as promising therapeutic drugs for MDMA abuse.

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

Background: Bioavailable testosterone (BT) is measured [assayed BT (aBT)] or calculated (cBT) in the diagnosis of hypogonadism in men. The cBT depends, however, on the values of the association constants of total testosterone (TT) for sex hormone-binding globulin (SHBG; K(s)) and albumin (K(a)), and its use therefore remains controversial., Methods: In 503 selected, untreated healthy men, 20-74 years old, we measured TT, dihydrotestosterone (DHT), and androstenediol (5-diol) by GC-MS, SHBG by RIA, and BT after ammonium sulfate precipitation or by calculation according to the law of mass action., Results: A slight decrease in TT, significant decreases in BT and 5-diol, no variation in DHT, and an increase in SHBG were observed with age. In young males (< or = 39 years), the lower normal limits were between 2.30 and 2.72 nmol/L for aBT and 8.50 nmol/L for TT. For K(s) = 1 x 10(9) L/mol and K(a) = 3.6 x 10(4) L/mol, the lower cBT limit was found to be 2-fold higher than for aBT. With optimized K(s) = 1.9 x 10(9) L/mol and K(a) = 2.45 x 10(4) L/mol, cBT values close to aBT were obtained. When 5-diol was included in the model as a competitive SHBG inhibitor, the correlation between cBT and aBT was better and the cBT:aBT ratios vs 5-diol were less biased., Conclusion: Lower normal serum aBT concentration in normal men appears to be between 2.30 and 2.72 nmol/L. Much higher serum cBT concentrations are associated with use of different association constants that may be inappropriate. When using the optimized binding constants, taking age-related 5-diol values into consideration slightly improves prediction of cBT.

Compelling evidence suggests that serotonin (5-HT) is necessary for the refined organization of the cerebral cortex. Here we sought to analyse the short- and long-term consequences of embryonic 5-HT depletion on the development of the cerebral neocortex of the rat. We focused on the migration and differentiation of the pyramidal (projection) and nonpyramidal (interneuron) neuronal populations. Our paradigm used daily injection of DL-P-chlorophenylalanine (PCPA), a reversible inhibitor of 5-HT synthesis, during the E12-17 stage of embryonic development, when major events in corticogenesis take place. We monitored the 5-HT depletion induced by this treatment and showed that it led to subtle alterations in both the pyramidal and nonpyramidal neuronal populations. We found that E12-17 PCPA treatment altered the maturation of pyramidal neurons of layers III and V of the somatosensory cortex, with these cells displaying reduced dendritic arborization and complexity. These long-lasting alterations were not associated with modification of cortical BDNF levels at postnatal stages. We also showed that PCPA treatment transiently altered the incorporation in the cortical plate of interneurons derived from the caudal ganglionic eminence, and persistently affected the differentiation of a subpopulation expressing calretinin and/or cholecystokinin.

Serotonin 5-HT(2B) receptors are often coexpressed with 5-HT(1B) receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT(1B) and 5-HT(2B) receptor cross-talk have not been elucidated. We hypothesized that 5-HT(2B) and 5-HT(1B) receptors each affect the others' signaling by modulating the others' trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT(2B) and 5-HT(1B) receptors when expressed alone and upon coexpression in LMTK(-) murine fibroblasts. Time-lapse confocal microscopy and whole-cell radioligand binding analyses revealed that, when expressed alone, 5-HT(2B) and 5-HT(1B) receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT(2B) receptors was accelerated 5-fold and was insensitive to a 5-HT(2B) receptor antagonist. In this context, 5-HT(2B) receptors did internalize in response to a 5-HT(1B) receptor agonist. In contrast, co-expression did not render 5-HT(1B) receptor internalization sensitive to a 5-HT(2B) receptor agonist. The altered internalization kinetics of both receptors upon coexpression was probably not due to direct interaction because only low levels of colocalization were observed. Antibody knockdown experiments revealed that internalization of 5-HT(1B) receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT(2B) receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT(2B) receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT(1B) receptor internalization became entirely Caveolin1-independent in a protein kinase Cepsilon-dependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT(1B) and 5-HT(2B) receptors influences the internalization pathways and kinetics of both receptors.

Wilson disease (WD) is an autosomal recessive disorder of copper biliary excretion caused by an impaired function of ATP7B, a metal-transporting P-type ATPase encoded by WD gene. It results in copper accumulation, mostly in liver and brain tissues. Mutation analysis was carried out on 11 WD French unrelated patients presenting a predominant neurological form of this illness. SSCP and dHPLC analysis followed by sequencing of the 21 exons and their flanking introns were performed. Thirteen different mutations in a total of 17, and, among them, 10 novel variants were evidenced. Two deletions (c.654&#95;655delCC and c.1745&#95;1746delTA), 4 missense mutations (p.F763Y, p.G843R, p.D918A and p.L979Q), 1 nonsense mutation (p.Q1200X), 1 splice site mutation (c.1947-1G>C) and 2 intronic silent substitutions (c.2448-25G>T and c.3412+13T>A) were detected. These data extend the mutational spectrum of the disease, already known to be a very heterogeneous genetic disorder. As compared to hepatic manifestations, the phenotypes associated to these mutations confirm that neurological presentations associated with other mutations than p.H1069Q are also often late in their onset. Most of these neurological forms probably correspond to an attenuated impairment of copper metabolism, as compared to hepatic forms of the disease, mostly diagnosed earlier.

We investigated a possible beneficial role for bilirubin, one of the products of heme degradation by the cytoprotective enzyme heme oxygenase-1 in counteracting Escherichia coli endotoxin-mediated toxicity. Homozygous jaundice Gunn rats, which display high plasma bilirubin levels due to deficiency of glucuronyl transferase activity, and Sprague-Dawley rats subjected to sustained exogenous bilirubin administration were more resistant to endotoxin (LPS)-induced hypotension and death compared with nonhyperbilirubinemic rats. LPS-stimulated production of nitric oxide (NO) was significantly decreased in hyperbilirubinemic rats compared with normal animals; this effect was associated with reduction of inducible NO synthase (NOS2) expression in renal, myocardial, and aortic tissues. Furthermore, NOS2 protein expression and activity were reduced in murine macrophages stimulated with LPS and preincubated with bilirubin at concentrations similar to that found in the serum of hyperbilirubinemic animals. This effect was secondary to inhibition of NAD(P)H oxidase since 1) inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS, 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro, and 3) down-regulation of NOS2 by bilirubin was reversed by addition of NAD(P)H. These findings indicate that bilirubin can act as an effective agent to reduce mortality and counteract hypotension elicited by endotoxin through mechanisms involving a decreased NOS2 induction secondary to inhibition of NAD(P)H oxidase.

Although compelling evidence has shown that obsessive-compulsive disorder (OCD) has a strong genetic component, its genetic basis remains to be elucidated. Identifying biological abnormalities in nonaffected relatives is one of the strategies advocated to isolate genetic vulnerability factors in complex disorders. Since peripheral serotonergic disturbances are frequently observed in OCD patients, the aim of this study was to investigate if they could represent endophenotypes, by searching for similar abnormalities in the unaffected parents of OCD patients. We assessed whole blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor-binding characteristics, and platelet inositol trisphosphate (IP3) content in a sample of OCD probands (n = 48) and their unaffected parents (n = 65), and compared them with sex- and age-matched controls (n = 113). Lower whole blood 5-HT concentration, fewer platelet 5-HTT-binding sites, and higher platelet IP3 content were found in OCD probands and their unaffected parents compared to controls. Whole blood 5-HT concentration showed a strong correlation within families (p < 0.001). The only parameter that appeared to discriminate affected and unaffected subjects was 5-HT2A receptor-binding characteristics, with increased receptor number and affinity in parents and no change in OCD probands. The presence of peripheral serotonergic abnormalities in OCD patients and their unaffected parents supports a familial origin of these disturbances. These alterations may serve as endophenotypic markers in OCD, and could contribute to the study of the biological mechanisms and genetic underpinnings of the disorder.

Although potentially implicated in several physiological functions, few functional mutations have been identified in the human 5-hydroxytryptamine (HT)(2B) receptor gene. A heterozygous mutation R393X in the 5-HT(2B) receptor was recently identified in a patient diagnosed with pulmonary hypertension after intake of the anorexigenic dexfenfluramine. Although reported to generate a lack of function, this C terminus-truncated 5-HT(2B) receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X-mutated 5-HT(2B) receptor. In stably transfected cells, we confirmed the loss of inositol 1,4,5-trisphosphate stimulation caused by the G(alphaq) uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric-oxide synthase stimulation. However, the truncated R393X receptor presented 1) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, 2) a preferential coupling to G(alpha13) as shown by blocking antiserum, and 3) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT(2B) receptor, the C terminus, including the palmitoylation and phosphorylation sites, is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C terminus can generate a switch of coupling to G(alpha13), a reduced NO synthase activation, and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction.

Background: The serotonergic 5-HT2B receptor regulates cardiomyocyte development and growth. A putative contribution of this receptor to fibroblast-dependent cardiac function has not been identified., Methods and Results: By mimicking sympathetic stimulation with chronic isoproterenol perfusion in vivo, we found that mice developed a cardiac hypertrophy, which was prevented by exposure to the 5-HT2B receptor antagonists SB206553 or SB215505 or in 5-HT2B receptor-knockout mice. The isoproterenol-induced hypertrophy was associated with an increase in the plasma levels of interleukin-1beta and tumor necrosis factor-alpha but not interleukin-6. In contrast, the plasma isoproterenol-induced cytokine increase was not observed in either 5-HT2B receptor-mutant or wild-type mice perfused with isoproterenol+SB206553. We demonstrated that stimulation of wild-type cardiac fibroblasts by isoproterenol markedly increased the production of the interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokines. Strikingly, we found that this isoproterenol-induced cytokine production was abolished by SB206553 or in 5-HT2B receptor-knockout fibroblasts. Serotonin also stimulated production of the 3 cytokines in wild-type fibroblasts, which was effectively reduced in 5-HT2B receptor-knockout fibroblasts., Conclusions: Our results demonstrate for the first time that 5-HT2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by cardiac fibroblasts.

Objective: Serotonin reuptake inhibitors (SRIs) are the most efficient pharmacological treatment of obsessive-compulsive disorders (OCD). Previous studies have suggested that some peripheral serotonergic parameters can be used to predict the clinical outcome of the treatment of OCD patients with SRIs. We tried to identify further peripheral serotonergic parameters that could help predict the clinical outcome of SRI treatment in a sample of patients with OCD., Methods: We compared 19 OCD patients before and after 8 weeks of SRI treatment with 19 sex-matched and age-matched controls. We assessed clinical improvement and whole-blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor binding characteristics and platelet IP3 content., Results: Before treatment, OCD patients had higher platelet IP3 content and fewer 5-HTT binding sites than the controls. Treatment with SRIs further lowered the number of 5-HTT binding sites, normalized platelet IP3 contents, and lowered the number of platelet 5-HT2A binding sites and whole-blood 5-HT concentrations below control values. The patients who improved most following SRI treatment had higher whole-blood 5-HT concentrations before treatment., Conclusion: Our results confirm that whole-blood 5-HT concentration is a predictor for clinical improvement and indicate that abnormal intracellular mechanisms may be involved in OCD patients, in particular, the overstimulation of the phosphoinositide signaling system.

Behavioral analyses of mice intoxicated by the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) have generated conflicting results. We therefore analyzed the relationship between behavioral changes, loss of monoamine levels, and loss of dopaminergic cell bodies in groups of mice intoxicated with acute or subchronic MPTP protocols. Despite a higher degree of neuronal loss in the mice intoxicated using subchronic protocols, dopamine loss was severe and homogeneous in the striatum in all groups. Dopamine levels were less severely reduced in the frontal cortex in the three groups of MPTP-intoxicated mice. Norepinephrine and serotonin levels in the striatum were decreased only in the mice intoxicated with the acute protocol. The most surprising result was that the mice intoxicated with the subchronic protocols were more active than the saline-treated mice. As reported in rats with dopamine depletion in the prefrontal cortex, the hyperactivity observed in our mice could be due to the reduced dopamine levels detected in this structure.

Objective: Esmolol, an ultra-short-acting beta-blocker, is known to attenuate myocardial ischemia-reperfusion injury. The aim of this study was to compare the effects of esmolol and potassium on myocardial metabolism during continuous normothermic retrograde blood cardioplegia., Methods: Forty-one patients operated on for isolated aortic valve stenosis were randomly assigned to continuous coronary infusion with either potassium or esmolol during cardiopulmonary bypass. Myocardial metabolism was assessed by measuring the transmyocardial gradient of oxygen content indexed to left ventricular mass of glucose, lactate, and nitric oxide. To do so, blood samples were simultaneously withdrawn upstream (in the cardioplegia line) and downstream of the myocardium (in the left coronary ostium) 10 and 30 minutes after aortic crossclamping., Results: Although the cardioplegia flow rate and pressure were similar, esmolol markedly reduced the transmyocardial gradient of oxygen content indexed to left ventricular mass compared with potassium: 13 +/- 6 vs 20 +/- 6 mL of oxygen per liter of blood per 100 g of myocardium, respectively, at 10 minutes and 16 +/- 8 vs 24 +/- 8 mL of oxygen per liter of blood per 100 g of myocardium, respectively, at 30 minutes (P =.009). Coronary glucose and lactate transmyocardial gradients were similar in both groups, indicating adequate myocardial perfusion in all patients at all times. In addition, during retrograde cardioplegia, esmolol showed a lower nitric oxide release compared with that caused by potassium (39 +/- 49 micro mol x L(-1) for potassium vs 14 +/- 8 micro mol x L(-1) for esmolol at 10 minutes and 39 +/- 47 micro mol x L(-1) for potassium vs 6 +/- 8 micro mol x L(-1) for esmolol at 30 minutes, P =.05). However, hemodynamic parameters and plasma troponin I levels remained unchanged postoperatively between the 2 types of cardioplegia., Conclusion: Esmolol provides potent myocardial protection in hypertrophied hearts, at least in part, by reducing myocardial oxygen metabolism.

Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity.

The loss of dopaminergic neurons in Parkinson's disease is associated with a glial reaction and the overproduction of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). TNF-alpha acts via two different receptors, TNFR1 and TNFR2, and is believed to have both a neuroprotective and a deleterious role for neurons. In order to analyze the putative role of TNF-alpha in parkinsonism, we compared the effect of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice lacking TNFR1, TNFR2, or both receptors and in wild-type littermates. We show that MPTP does not affect spontaneous activity or anxiety in any of the groups and that it reduces motor activity on a rotarod in double knock out mice but not in mice lacking only one receptor. Postmortem analysis revealed no differences in the number of nigral dopaminergic neurons whatever the group. In contrast, striatal dopamine level was slightly decreased in double knock-out mice and more reduced by MPTP in this group than in the other groups of mice. In addition, dopamine turnover was significantly more increased in double knock out mice after MPTP injection. These data suggest that TNF-alpha does not participate in the death of dopaminergic neurons in parkinsonism but that it slightly alters dopamine metabolism or the survival of dopaminergic terminals by a mechanism involving both receptors.

We examined the effect of pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist of the thiazolidinedione class, on dopaminergic nerve cell death and glial activation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. The acute intoxication of C57BL/6 mice with MPTP led to nigrostriatal injury, as determined by tyrosine hydroxylase (TH) immunocytochemistry, and HPLC detection of striatal dopamine and metabolites. Damage to the nigrostriatal dopamine system was accompanied by a transient activation of microglia, as determined by macrophage antigen-1 (Mac-1) and inducible nitric oxide synthase (iNOS) immunoreactivity, and a prolonged astrocytic response. Orally administered pioglitazone (approximately 20 mg/kg/day) attenuated the MPTP-induced glial activation and prevented the dopaminergic cell loss in the substantia nigra pars compacta (SNpc). In contrast, there was little reduction of MPTP-induced dopamine depletion, with no detectable effect on loss of TH immunoreactivity and glial response in the striatum of pioglitazone-treated animals. Low levels of PPARgamma expression were detected in the ventral mesencephalon and striatum, and were unaffected by MPTP or pioglitazone treatment. Since pioglitazone affects primarily the SNpc in our model, different PPARgamma-independent mechanisms may regulate glial activation in the dopaminergic terminals compared with the dopaminergic cell bodies after acute MPTP intoxication.

Purpose: To observe the morphology and physiology of the retina in rats 11 weeks after a constant (24-hour) but moderate (500-lux) illumination for 1 week., Methods: Levels of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, and taurine were measured by high-pressure liquid chromatography (HPLC) in the retina and vitreous humor of albino (Wistar) and pigmented (Long-Evans) rats. Semithin sections were used to determine retinal morphology. The TUNEL method was used to detect cells degenerating by apoptosis. Because the GABAergic system has been shown to be particularly sensitive to the loss of photoreceptors, an additional immunohistochemical study using anti-GABA, anti-glutamate decarboxylase (GAD)(67) and anti-GAD(65) antibodies was performed., Results: No apparent morphologic changes were found in the retina of pigmented rats after constant illumination, whereas in albino rats disappearance of photoreceptors (except in the extreme retinal periphery) and cell bodies was observed. A significant number of TUNEL-positive nuclei also occurred in the remaining nuclear and ganglion cell layers. However, no change in the distribution of GABA, GAD(67), and GAD(65) immunoreactivities was found in either strain under constant illumination compared with control animals. Constant illumination affected the retinal levels of aspartate, glutamate, glutamine, glycine in both strains, whereas GABA contents did not change and taurine was decreased only in albino rats. A significant increase of vitreal glutamate levels was also found in both strains and of taurine levels only in albino rats., Conclusions: Phototoxicity can provoke durable retinal alterations beyond the period of lighting, suggesting progressive and probably continuous modifications of retinal physiology, even in pigmented animals in which the retina seems morphologically normal.

Pharmacological evidence supports a role of a transient decreased endogenous nitric oxide (NO) synthesis in ovalbumin (OVA)-induced early airway hyperresponsiveness in guinea pigs. However, no data are available regarding the expression and activity of the constitutive NO synthases (cNOS; NOS1 and NOS3, nNOS and eNOS, respectively) in this model. Therefore, we evaluated cNOS activity (conversion of L-[3H]arginine to L-[3H]citrulline in the presence of Ca2+ and calmodulin), nitrate and nitrite (NOx) concentration (modified Griess method), and NOS1 and NOS3 protein expression (Western blot) in lung homogenates and in the tracheal smooth muscle from OVA-immunized and multiple aerosol-challenged guinea pigs (six challenges, once daily). The expression and activity of the inducible NOS isoform (NOS2), the levels of exhaled NO, and the in vivo airway reactivity were also determined. Constitutive NOS activity and NO(x) concentration were significantly lower 6 h after the last OVA challenge as compared with saline exposure, being similar at 24 h. Expression of NOS1 paralleled cNOS activity, which was reduced 6, but not 24 h after OVA challenge. The decrease in NOS1 expression was accompanied by a significant decrease in the amounts of exhaled NO and by a maximal airway hyperresponsiveness to histamine. The levels of NOS3 were not modified at the two time points evaluated, and no NOS2 expression and activity were found at any time point. Similar modifications were observed in the tracheal smooth muscle. We conclude that OVA stimulation in immunized guinea pigs induced a transient reduction in NOS1 protein expression and activity in the respiratory system, which probably participates in airway hyperresponsiveness.

Objective: We investigated the expression in rabbit basilar arteries of cyclooxygenase (COX)-2, which is the inducible isoform of the enzyme of prostaglandin (PG) production, and the concentrations of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and representative PGs in the cerebrospinal fluid (CSF) after experimental subarachnoid hemorrhage (SAH)., Methods: Seven sets of basilar arteries were removed from control rabbits and from rabbits killed 1 and 3 days after induced SAH. The arteries were subjected to identical simultaneous immunolabeling for examination with a confocal microscope. One-half of each artery was stained for the constitutive form COX-1 and the other half for COX-2. CSF was sampled in control animals and at 6 hours, 1 day, and 3 days for assays of TNFalpha, PGE2, and 6-keto-PGF1 (metabolite of PGI2)., Results: COX-1 immunoreactivity in the endothelial layer was similar in the three groups. Weak endothelial COX-2 immunoreactivity was found in arteries of control animals. COX-2 staining was higher in the group killed at 3 days compared with the control group (P < 0.05). The levels of PGE2 and 6-keto-PGF1alpha in the CSF peaked significantly at 6 hours, then decreased at 3 days to the basal level (PGE2) or significantly lower (6-keto-PGF1). TNFalpha was undetectable in control CSF, significantly higher (P < 0.001) at 6 hours, and undetectable at 3 days., Conclusion: After SAH, endothelial COX-1 immunoreactivity does not change, whereas overexpression of COX-2 occurs at 3 days. This induction does not seem linked to TNFalpha production, nor is it responsible for early raised levels of PGE2 and PGI2 in the CSF. We suggest that the role of induced COX-2 may be to modify gene expression and hence alter the properties of the vessel wall after SAH.

Migraine is one of the few pathologies which gave rise to a tremendous number of physiopathological hypotheses. The variability of its clinical features and of the crisis initiating triggers, together with the numerous functional and/or biological abnormalities reported in migrainous patients, led to multiple 'theories' about migraine. For instance, migraine attacks may be associated with modifications of cerebral blood flow, and/or alterations at the cellular (neuronal and peripheral: platelets, mast cells, etc.) and subcellular (mainly mitochondrial) levels leading to variations of parameters such as serotonin, vasoactive neuropeptides, histamine, nitric oxide, neuroactive amino acids, etc. However, these modifications are mainly related to migraine attacks but not to migrainous patients. These emphasize how important is the distinction between the crisis mechanism(s) and the determinism of migraine illness. Despite the absence of any true animal model of migraine attack, the obtention, through the activation of the trigemino-vascular complex, of an experimental meningeal neurogenic inflammation was a clear breakthrough for the understanding of the migraine attack. Concerning the determinism of migraine, its familial characteristic has been known for a long time, but genetic studies started only recently. Despite some important contributions, the respective roles of genetic and environmental factors, as well as the transmission mode of migraine, remain largely to be determined. Practically, these genetic data, which really concern only a very peculiar form of migraine--the familial hemiplegic one--do not have presently any diagnostic or therapeutic application.

The cytokine network and infection severity were characterized during disseminated cryptococcosis in tumor necrosis factor (TNF)- and lymphotoxin (Lt)-alpha-deficient mice. On day 16, the fungus burden was higher and median survival time was reduced, as was polymorphonuclear leukocyte infiltrate in the brains of knockout mice. TNF/Lt-alpha-deficient mice had lower levels of interleukin (IL)-6 in lungs and brains, IL-1beta, and the chemokine KC in brain and spleen and of the chemokine monocyte chemoattractant protein (MCP)-1 in spleen than control animals. In contrast, higher levels of IL-6, IL-10, and MCP-1 in plasma and higher levels of IL-12, interferon (IFN)-gamma, and nitrite/nitrate were found in all compartments of TNF/Lt-alpha-deficient mice. These data confirm that TNF or Lt-alpha is a key cytokine for the anticryptococcal response and demonstrate its major role for the induction of IL-1beta, IL-6, and KC in the brain; however, its presence is not a prerequisite for IL-12, IFN-gamma, and nitrite/nitrate production.

Objective: Growth regulatory properties of nitric oxide (NO) in cultured endothelial cells is controversial. The aim of our study was to investigate the effect of L-arginine, the endogenous NO precursor, and L-NAME, an inhibitor of NO synthase on the reendothelialization process after angioplasty., Methods: Fifty-five New Zealand White rabbits underwent denudation of the left iliac artery. After injury the rabbits were randomized in three groups: L-arginine 2.25% (L-arginine, n = 19); NG-nitro-L-arginine methyl ester 15 mg/kg/day (L-NAME, n = 19); and placebo (controls, n = 17). Treatment was solubilized in drinking water. Reendothelialization was evaluated at 4 weeks by macroscopic evaluation of Evans blue staining and endothelial-specific immunostaining (CD-31) on cross sections. Intimal hyperplasia was evaluated by morphometric analysis., Results: Despite a significant increase in plasma arginine (P = 0.001) and a reduction in intimal hyperplasia (P = 0.003) with L-arginine, neither agent had a significant effect on reendothelialization at 4 weeks (controls = 36 +/- 4%, L-arginine = 43 +/- 3%, L-NAME = 33 +/- 4%; NS)., Conclusion: These results suggest that, in spite of previously demonstrated effects on neointimal hyperplasia, the NO pathway does not influence the regrowth of macrovascular endothelial cells in vivo.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

The recently characterized 5-HT2B subfamily of serotonin receptors has now been reported from three different species: human, rat and mouse. Their genomic structures include 2 introns present at identical positions. Despite this similarity, their respective protein sequences show some diversities. In addition, the pharmacology of these receptors is distantly related, and their sites of expression vary amongst species. Thus, it appears difficult at present to unambiguously classify these receptors into the same subfamily, raising the possibility of the existence of other 5-HT2B-like receptors, yet to be discovered.

Using a microdialysis technique we showed that the exposure of the rat striatum to glutamate yields hydroxyl radicals and results in striatal damage. We postulated that dopamine release is enhanced by glutamate perfusion and that the enzymatic metabolism of dopamine may account for this hydroxyl radical formation. The inhibition of monoamine oxidases by i.p. co-administration of clorgy-line and deprenyl reduced hydroxyl radical production induced by glutamate perfusion, but significantly increased the striatal damage. Our results suggest that the enzymatic metabolism of dopamine participates in glutamate-induced hydroxyl radical generation but that other by-products of dopamine may be responsible for the aggravation of the striatal injury.

Background/aims: In the gastric mucosa of Helicobacter pylori-infected subjects, we previously detected N alpha-methyl histamine (N alpha-MeHA), a minor catabolite of histamine and a potent agonist of histamine H3 receptors. The origin of N alpha-MeHA and its effects on gastric histamine and somatostatin in infected subjects were investigated., Methods: Ten noninfected patients and 13 patients with intense colonization were compared. N alpha-MeHA content and its synthetic enzyme activity, N alpha-histamine methyltransferase, binding of [3H]N alpha-MeHA, histamine and somatostatin contents, and histidine decarboxylase activity were assayed in antral and fundic biopsy specimens and in cultured H. pylori strains., Results: Gastric histamine and somatostatin contents as well as histidine decarboxylase activity were decreased in infected patients and were restored to normal after antimicrobial treatment. Both N alpha-MeHA and N alpha-histamine methyltransferase activity were present in the mucosa of infected patients and in cultured strains and were very low in noninfected patients or after eradication of H. pylori. [3H]N alpha-MeHA bound to gastric mucosa but not to cultured strains. The [3H]N alpha-MeHA specific binding sites were characterized as H3 receptors. The amount of bound [3H]N alpha-MeHA seemed correlated positively with somatostatin content and histidine decarboxylase activity and negatively with N alpha-MeHA content and N alpha-histamine methyltransferase activity., Conclusions: H. pylori is the main source of gastric N alpha-MeHA that may lower histidine decarboxylase activity and somatostatin content through H3 receptors.

Brain microdialysis was used to study changes in the glutamate and aspartate extracellular concentrations in the striatum of conscious rats submitted to 30 minutes cerebral ischaemia, using the four-vessel occlusion model. Perfusion of the N-methyl-D-aspartate (NMDA) receptor channel blockers, dizocilpine (MK-801; 75 microM) and Mg2+ (2.5 mM), inhibited the ischaemia-induced accumulation of glutamate and aspartate. The AMPA/kainate receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamylbenzo (F) quinoxaline (NBQX; 15 microM and 450 microM) had no effect on glutamate and aspartate levels during ischaemia. On the other hand, omission of Ca2+ from the perfusing solution did not alter the increases in glutamate and aspartate induced by ischaemia. These results suggest that the glutamate and aspartate accumulation in four-vessel occlusion ischaemia is mediated by activation of NMDA receptors in a Ca2+ independent manner.

Rabbit leptomeningeal arteries contain granular cells resembling mast cells that frequently contact autonomic and sensory nerve profiles. In the present in vitro study, we determined whether these cells could be stimulated by substance P (SP) and calcitonin gene-related peptide (CGRP), which are stored and released by sensory C fibers. Immunohistochemistry of the middle cerebral artery showed that 5-HT was stored only in mast cell-like granules. This pool of 5-HT decreased in a dose-dependent manner when exogenous SP and CGRP were added to the incubation solution or when endogenous neuropeptides were released from nerve terminals by capsaicin. The simultaneous administration of CGRP and SP induced a dramatic exocytosis and a 5-HT release significantly greater than the sum of the individual effects of the two neuropeptides. We conclude that, as in classical connective tissue mast cells, the amine content of these granular cells can be released by a degranulation process induced by neuropeptides. The effects of capsaicin suggest that this phenomenon can be triggered by axon reflex of C fibers. The data also provide the first evidence of a synergistic action of SP and CGRP on mast cell degranulation.

Serotoninergic mechanisms are thought to play a role in portal hypertension. Because this biomine is metabolized by the liver, peripheral blood and plasma levels of 5-hydroxytryptamine and 5-hydroxyindole acetic acid (the main metabolite of 5-hydroxytryptamine) were measured in 30 patients with cirrhosis. Whole-blood 5-hydroxytryptamine levels were significantly lower in patients with cirrhosis (158 +/- 28 nM) than in age-matched controls (332 +/- 19 nM), and no correlation was found between these levels and the severity of cirrhosis. Unconjugated plasma 5-hydroxytryptamine levels, an indication of the active form of 5-hydroxytryptamine, were significantly higher in patients with cirrhosis than in controls (6.8 +/- 1.7 nM and 3.4 +/- 0.5 nM, respectively), and in patients with cirrhosis these levels were higher in Pugh grade A than in Pugh grade C patients. Conjugated-plasma 5-hydroxytryptamine levels were not significantly different between patients with cirrhosis (32.2 +/- 8.1 nmol/L) and controls (16.4 +/- 1.4 nmol/L). Plasma 5-hydroxyindole acetic acid was significantly lower in patients with cirrhosis than in controls (1.5 +/- 0.1 nmol/L and 2.3 +/- 0.1 nmol/L, respectively). In conclusion, this study shows that serotoninergic mechanisms are altered in patients with cirrhosis.

Spontaneous and all-trans-retinoic acid (RA)-induced differentiation of normal human monocytes and of leukemic THP-1 monocytes into macrophages resulted in a progressive loss of adenosine 3',5'-cyclic monophosphate production induced by histamine via typical H2 receptors (H2R). In THP-1 cells and in HL-60 human acute myelocytic leukemia cells, RA treatment increased the abundance of the 4.5-kb messenger RNA of the H2R gene fourfold, suggesting transcriptional control by a RA response element. Scatchard plots of [3H]tiotidine binding indicated the expression of H2R with similar affinity and binding capacity in THP-1 monocytes and macrophages, while the conversion of normal monocytes into macrophages decreased H2R density from 91.8 to 43.1 fmol/mg protein, with no change in affinity (Kd = 9.9 to 11.2 nM). In THP-1 macrophages, histamine inhibited 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced H2O2 formation via the activation of H2 receptors. Expression of the H2R gene, histamine accumulation, and histidine decarboxylase activity were also demonstrated in normal human monocytes/macrophages and peripheral lymphocytes. Histamine and H2R may therefore affect, via intracrine, autocrine, and paracrine pathways, various immune and inflammatory responses of the lymphoid and myeloid progenitors and lineages in the bone marrow and peripheral tissues.