Theiler's Murine Encephalomyelitis Virus Replicates in Primary Neuron Cultures and Impairs Spine Density Formation.

In this study, we examined infection with the highly neurovirulent GDVII, the less neurovirulent DA strains, and with a mutant DA, which lacks the L* protein (L*-1) involved in viral persistence and demyelinating disease, to analyze the direct effects of Theiler's murine encephalomyelitis virus (TMEV) replication using primary cultures of mouse brain hippocampal neurons. All viruses replicate in cultured neurons, with GDVII having the highest titers and L*-1 the lowest. Accordingly, all were positive for viral antigen staining 3 days postinfection (dpi), and DA and L*-1 were also positive after 12 dpi. NeuN + immunostaining showed an early and almost complete absence of positive cells in cultures infected with GDVII, an approximately 50% reduction in cultures infected with DA, and fewer changes in L*-1 strains at 3 dpi. Accordingly, staining with chloromethyltetramethylrosamine orange (Mitotracker OrangeTM) as a parameter for cell viability showed similar results. Moreover, at 1 dpi, the strain DA induced higher transcript levels of neuroprotective genes such as IFN-Iß, IRF7, and IRF8. At 3 dpi, strains GDVII and DA, but not the L*-1 mutant, showed lower PKR expression. In addition, confocal analysis showed that L*-1-infected neurons exhibited a decrease in spine density. Treatment with poly (I:C), which is structurally related to dsRNA and is known to trigger IFN type I synthesis, reduced spine density even more. These results confirmed the use of mouse hippocampal neuron cultures as a model to study neuronal responses after TMEV infection, particularly in the formation of spine density.

Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons.

The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.

Neuronal Glycoprotein M6a: An Emerging Molecule in Chemical Synapse Formation and Dysfunction.

The cellular and molecular mechanisms underlying neuropsychiatric and neurodevelopmental disorders show that most of them can be categorized as synaptopathies-or damage of synaptic function and plasticity. Synaptic formation and maintenance are orchestrated by protein complexes that are in turn regulated in space and time during neuronal development allowing synaptic plasticity. However, the exact mechanisms by which these processes are managed remain unknown. Large-scale genomic and proteomic projects led to the discovery of new molecules and their associated variants as disease risk factors. Neuronal glycoprotein M6a, encoded by the GPM6A gene is emerging as one of these molecules. M6a has been involved in neuron development and synapse formation and plasticity, and was also recently proposed as a gene-target in various neuropsychiatric disorders where it could also be used as a biomarker. In this review, we provide an overview of the structure and molecular mechanisms by which glycoprotein M6a participates in synapse formation and maintenance. We also review evidence collected from patients carrying mutations in the GPM6A gene; animal models, and in vitro studies that together emphasize the relevance of M6a, particularly in synapses and in neurological conditions.

Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity.

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.

Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS.

Nowadays, great efforts are made to gain insight into the molecular mechanisms that underlie structural neuronal plasticity. Moreover, the identification of signaling pathways involved in the development of psychiatric disorders aids the screening of possible therapeutic targets. Genetic variations or alterations in GPM6A expression are linked to neurological disorders such as schizophrenia, depression, and Alzheimer's disease. GPM6A encodes the neuronal surface glycoprotein M6a that promotes filopodia/spine, dendrite, and synapse formation by unknown mechanisms. A substantial body of evidence suggests that the extracellular loops of M6a command its function. However, the proteins that associate with them and that modulate neuronal plasticity have not been determined yet. To address this question, we generated a chimera protein that only contains the extracellular loops of M6a and performed a co-immunoprecipitation with rat hippocampus samples followed by TMT/MS. Here, we report 72 proteins, which are good candidates to interact with M6a's extracellular loops and modify its function. Gene ontology (GO) analysis showed that 63% of the potential M6a's interactor proteins belong to the category "synapse," at both sides of the synaptic cleft, "neuron projections" (51%) and "presynapse" (49%). In this sense, we showed that endogenous M6a interacts with piccolo, synaptic vesicle protein 2B, and synapsin 1 in mature cultured hippocampal neurons. Interestingly, about 28% of the proteins left were related to the "myelin sheath" annotation, suggesting that M6a could interact with proteins at the surface of oligodendrocytes. Indeed, we demonstrated the (cis and trans) interaction between M6a and proteolipid protein (PLP) in neuroblastoma N2a cells. Finally, the 72 proteins were subjected to disease-associated genes and variants screening by DisGeNET. Apart from the diseases that have already been associated with M6a, most of the proteins are also involved in "autistic disorder," "epilepsy," and "seizures" increasing the spectrum of disorders in which M6a could play a role. Data are available via ProteomeXchange with identifier PXD017347.

The Membrane Glycoprotein M6a Endocytic/Recycling Pathway Involves Clathrin-Mediated Endocytosis and Affects Neuronal Synapses.

Single point mutations or variations in the expression of the gene encoding the neuronal glycoprotein M6a have been associated with psychiatric disorders such as Alzheimer's disease, depression and schizophrenia. In cultured neurons, M6a positively contributes to neurite extension, axon guidance, filopodia/spine outgrowth, and synapse formation. The endocytic processes of neuronal membrane proteins are linked to the differentiation, growth, signaling and plasticity of neurons. However, the roles of M6a and the precise mechanisms through which M6a internalizes and recycles back to the neuronal membrane are unknown. Here, by using a controlled in vitro assay, we showed that if 30-40% of M6a is endocytosed, the number of synapses in hippocampal neurons decreases. When re-establishing the levels of M6a at the cell surface, the number of synapses returned to normal values. M6a internalization involves clathrin-coated pits, probably by association between the adaptor protein 2 and the 251YEDI254 "tyrosine-based" motif located within the C-tail of M6a. Upon endocytosis, M6a is sorted to early endosome antigen 1- and Rab5-positive endosomes and then sorted back to the cell surface via Rab11-positive endosomes or to degradation via Rab7 and, finally LAMP-1-positive endosomes. Our results demonstrated that the levels of M6a at the cell surface modified the formation/maintenance of synapses, without altering the protein levels of synaptophysin or N-methyl-D-aspartate receptor type-1. This novel mechanism might be relevant during neuronal development, pruning and/or many of the neurological disorders in which the number of synapses is affected.

Evidence for a role of glycoprotein M6a in dendritic spine formation and synaptogenesis.

Neuronal glycoprotein M6a belongs to the tetraspan proteolipid protein (PLP) family. Mutations in GPM6A gene have been related to mental disorders like schizophrenia, bipolar disorders and claustrophobia. M6a is expressed mainly in neuronal cells of the central nervous system and it has been extensively related to neuronal plasticity. M6a induces neuritogenesis and axon/filopodium outgrowth; however its mechanism of action is still unresolved. We recently reported that the integrity of the transmembrane domains (TMDs) 2 and 4 are critical for M6a filopodia induction. There is also experimental data suggesting that M6a might be involved in synaptogenesis. In this regard, we have previously determined that M6a is involved in filopodia motility, a process that is described in the first step of the filopodial model for synaptogenesis. In this work we analyzed the possible involvement of M6a in synaptogenesis and spinogenesis, and evaluated the effect of two non-synonymous SNPs present in the coding region of TMD2-GPM6A in these processes. The results showed that endogenous M6a colocalized with both, pre-synaptic (synaptophysin) and post-synaptic (NMDA-R1), markers along of neuronal soma and dendrites. M6a-overexpressing neurons displayed an increased number of synaptophysin and NMDA-R1 puncta and, also, an increased number of colocalization puncta between both markers. Conversely, the number of synaptic puncta markers in neurons expressing nsSNP variants was similar to those of control neurons. Overexpression of M6a is accompanied by an increase in spine density, particularly in mature spines, as compared with neurons expressing mGFP or GPM6A nsSNP variants. Taken together, these results suggest that M6a contributes positively to spine and, likely, synapse formation.

Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains.

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress-regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a-null mice develop a claustrophobia-like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single-nucleotide polymorphisms (SNPs) in the non-coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self-interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a-induced filopodia formation in neurons. In silico analysis of three non-synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus-Merzbacher disease. In this work we analyzed the functional contribution of transmembrane domains (TMDs) of the neuronal membrane glycoprotein M6a. We determined that certain glycines present in TMD2 and TMD4 are critical for filopodia induction in neurons. In addition, three nsSNPs located in the coding region of TMD2 and TMD3 of GPM6A impair M6a function by affecting its stability, folding and dimer formation.

Tyrosine 251 at the C-terminus of neuronal glycoprotein M6a is critical for neurite outgrowth.

Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension.

Neuronal glycoprotein M6a induces filopodia formation via association with cholesterol-rich lipid rafts.

A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca(2+) transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-ß-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation.

Role of ammonia and nitric oxide in the decrease in plasma prolactin levels in prehepatic portal hypertensive male rats.

OBJECTIVES: Since very little is known about neuroendocrine changes that occur in portal-systemic hepatic encephalopathy, we studied plasma prolactin (PRL) levels and the involvement of hyperammonemia, nitric oxide (NO) and dopaminergic and adrenergic systems in the control of this hormone secretion in a male rat model of prehepatic portal hypertension (PH). METHODS: We conducted in vivo studies to determine plasma ammonia and PRL levels. Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), epinephrine and norepinephrine content in medial basal hypothalamus (MBH) and anterior pituitary (AP) were measured. In addition, NO synthase (NOS) activity and protein expression were evaluated in APs. In in vitro studies, the APs from intact rats were incubated with different doses of ammonia and PRL secretion was determined. In ex vivo studies, the APs from normal and PH rats were incubated in the presence of ammonia and/or a NOS inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME) and PRL secretion was determined. RESULTS: PH rats had a significant increase in plasma ammonia levels (p < 0.001) and a decrease in plasma PRL levels (p < 0.05). Neither DA nor DOPAC content or DOPAC/DA ratios were modified in both MBH and APs; however, we observed a significant increase in norepinephrine content in both MBH and AP (p < 0.001 and p < 0.05, respectively) and a significant increase in epinephrine in APs (p < 0.001). Moreover, PH produced an increase in NOS activity (p < 0.01) and NOS protein expression (p < 0.0001) in APs. The ammonia (100 microM) significantly reduced PRL secretion from APs in vitro (p < 0.05). The presence of L-NAME, an inhibitor of NOS, abrogated the inhibitory effect of ammonia on PRL secretion from APs from control and PH rats. CONCLUSIONS: We found that plasma PRL levels were decreased in PH rats probably due to the high ammonia levels. The central noradrenergic system could also mediate this decrease. Also, the increase in NOS activity and/or content in AP induced NO production that directly inhibited PRL secretion from the AP, without the participation of the dopaminergic system.

Activation of nitric oxide synthesis in human endothelial cells using nomegestrol acetate.

OBJECTIVE: Recent clinical trials indicate that synthetic progestins may be unexpectedly relevant for the development of cardiovascular disease. The aim of this study was to establish whether nomegestrol acetate induces signaling events in human endothelial cells that differ from those of other progestins, such as natural progesterone or medroxyprogesterone acetate. METHODS: We used human endothelial cells to study the action of nomegestrol acetate (either alone or in the presence of estradiol [E2]) on the synthesis of nitric oxide (NO) and on the activity or expression of endothelial nitric oxide synthase (eNOS). We compared the effects of nomegestrol acetate with those of progesterone or medroxyprogesterone acetate. In addition, we characterized the signaling events recruited by these compounds. RESULTS: Progesterone and nomegestrol acetate increase NO synthesis by transcriptional and nontranscriptional mechanisms, whereas medroxyprogesterone acetate lacks such effects. When used together with physiological E2 concentrations, progesterone and nomegestrol acetate do not interfere with (or even enhance) E2 effects, whereas medroxyprogesterone acetate impairs E2 signaling. A marked difference in the recruitment of mitogen-activated protein kinase and phosphatidylinositol-3 kinase explains the divergent effects of the three gestagens. CONCLUSION: Our findings show significant differences in the signal transduction pathways recruited by progesterone, nomegestrol acetate, and medroxyprogesterone acetate in human endothelial cells that may have relevant clinical implications.

Effects of dydrogesterone and of its stable metabolite, 20-alpha-dihydrodydrogesterone, on nitric oxide synthesis in human endothelial cells.

OBJECTIVE: To investigate the effects of P, medroxyprogesterone acetate (MPA), and dydrogesterone (DYD) and its metabolite, 20-alpha-dihydrodydrogesterone (DHD) on endothelial synthesis of nitric oxide (NO) and characterize the signaling events recruited by these compounds. The Women's Health Initiative trial reports an excess of heart disease in postmenopausal women receiving MPA. DESIGN: Cell culture. SETTING: Research laboratory. PATIENT(S): Human endothelial cells from umbilical vein. INTERVENTION(S): Treatments with P, MPA, DYD, or DHD. MAIN OUTCOME MEASURE(S): Measure of NO release, endothelial nitric oxide synthase (eNOS) activity and expression, and activation of ERK 1/2 and Akt. RESULT(S): The administration of DYD alone or in combination with estrogen to endothelial cells results in neutral effects on NO synthesis and on the activity and expression of eNOS. In parallel, the stable metabolite DHD acts similarly to natural P, enhancing the expression of eNOS and inducing rapid activation of the enzyme through the regulation of the ERK 1/2 mitogen-activated protein kinase cascade. 20-Alpha-dihydrodydrogesterone and P also potentiate eNOS induction by E2. On the contrary, MPA does not trigger eNOS enzymatic activation and decreases the extent of eNOS induction by E2. CONCLUSION(S): These findings support the concept that synthetic progestins act differently on vascular cells and that hormonal preparations may differ as to their cardiovascular effects.

Estrogen receptor alpha interacts with Galpha13 to drive actin remodeling and endothelial cell migration via the RhoA/Rho kinase/moesin pathway.

Sex steroids control cell movement and tissue organization; however, little is known of the involved mechanisms. This report describes the ongoing dynamic regulation by estrogen of the actin cytoskeleton and cell movement in human vascular endothelial cells that depends on rapid activation of the actin-regulatory protein moesin. Moesin activation is triggered by the interaction of the C-terminal portion of cell membrane estrogen receptor alpha with the G protein Galpha(13), leading to activation of the small GTPase RhoA and of the downstream effector Rho-associated kinase. The resulting phosphorylation of moesin on Thr(558) is the means of moesin's binding to actin and the remodeling of the actin cytoskeleton. This cascade of events ensues within minutes of estradiol administration and results in changes in cell morphology and to the development of specialized cell membrane structures such as ruffles and pseudopodia that are necessary for cell movement. These findings expand our knowledge of the basis of estrogen's effects on human cells, including the regulation of actin assembly, cell movement and migration. They highlight novel pathways of signal transduction of estrogen receptor alpha through nontranscriptional mechanisms. Furthermore, exposure of this estrogen receptor-dependent, nongenomic action of estrogen on human vascular endothelial cells is especially relevant to the present interest in the role of estrogen in cardiovascular protection.

Altered blood-brain barrier permeability in rats with prehepatic portal hypertension turns to normal when portal pressure is lowered.

AIM: To study the blood-brain barrier integrity in prehepatic portal hypertensive rats induced by partial portal vein ligation,at 14 and 40 d after ligation when portal pressure is spontaneously normalized. METHODS: Adult male Wistar rats were divided into four groups: Group I: Sham14d , sham operated; Group II: PH14d , portal vein stenosis; (both groups were used 14 d after surgery); Group III: Sham40d, Sham operated and Group IV: PH40d Portal vein stenosis (Groups II and IV used 40 d after surgery). Plasma ammonia,plasma and cerebrospinal fluid protein and liver enzymes concentrations were determined. Trypan and Evans blue dyes, systemically injected,were investigated in hippocampus to study blood-brain barrier integrity. Portal pressure was periodically recorded. RESULTS: Forty days after stricture, portal pressure was normalized, plasma ammonia was moderately high, and both dyes were absent in central nervous system parenchyma. All other parameters were reestablished. When portal pressure was normalized and ammonia level was lowered, but not normal, the altered integrity of blood-brain barrier becomes reestablished. CONCLUSION: The impairment of blood-brain barrier and subsequent normalization could be a mechanism involved in hepatic encephalopathy reversibility.Hemodynamic changes and ammonia could trigger blood-brain barrier alterations and its reestablishment.

Effects of rimonabant, a selective cannabinoid CB1 receptor antagonist, in a rat model of Parkinson's disease.

Recent evidence suggest that the blockade of cannabinoid CB1 receptors might be beneficial to alleviate motor inhibition typical of Parkinson's disease (PD). In the present study, we have explored the motor effects of rimonabant, a selective antagonist of CB1 receptors, in a rat model of PD generated by an intracerebroventricular injection of 6-hydroxydopamine. Compared with rats subjected to unilateral injection of this toxin in the medial forebrain bundle, this model allows nigral dopaminergic neurons be symmetrically affected. Dose-response studies with 6-hydroxydopamine revealed that the application of 200 microg per animal caused hypokinetic signs (decreased ambulatory activity, increased inactivity, and reduced motor coordination), which paralleled several signs of degeneration of nigrostriatal dopaminergic neurons (dopamine depletion in the caudate-putamen, and decreased mRNA levels for tyrosine hydroxylase and superoxide dismutase-1 and -2 in the substantia nigra). In these conditions, the degree of hypokinesia and dopaminergic degeneration may be considered moderate, comparable to the disturbances occurring in early and middle stages of PD in humans, a period that might be appropriate to test the effects of rimonabant. There is also degeneration of other dopaminergic pathways out of the basal ganglia, but this does not appear to interfere significantly with the hypokinetic profile of these rats. Higher doses of 6-hydroxydopamine elevated significantly animal mortality and lower doses failed in general to reproduce motor inhibition. Like other animal models of PD, these rats exhibited an increase in the density of CB(1) receptors in the substantia nigra, which is indicative of the expected overactivity of the cannabinoid transmission in this disease and supports the potential of CB1 receptor blockade to attenuate hypokinesia associated with nigral cell death. Thus, the injection of 0.1 mg/kg of rimonabant partially attenuated the hypokinesia shown by these animals with no effects in control rats, whereas higher doses (0.5-1.0 mg/kg) were not effective. We also found that the antihypokinetic effects of low doses of rimonabant did not influence the dopamine deficits of these animals, as well as it did not modify GABA or glutamate transmission in the caudate-putamen. In summary, rimonabant may have potential antihypokinetic activity in moderate parkinsonism at low doses, but this effect is not related to changes in dopaminergic, GABAergic, or glutamatergic transmission in the striatum. Therefore, the elucidation of the neurochemical substrate involved in this effect remains a major challenge for the future.

The inhibitory effect of anandamide on luteinizing hormone-releasing hormone secretion is reversed by estrogen.

Because Delta-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10(-9) M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.

Hyperammonemia, brain edema and blood-brain barrier alterations in prehepatic portal hypertensive rats and paracetamol intoxication.

AIM: To study the blood-brain barrier integrity, brain edema, animal behavior and ammonia plasma levels in prehepatic portal hypertensive rats with and without acute liver intoxication. METHODS: Adults male Wistar rats were divided into four groups. Group I: sham operation; II: Prehepatic portal hypertension, produced by partial portal vein ligation; III: Acetaminophen intoxication and IV: Prehepatic portal hypertension plus acetaminophen. Acetaminophen was administered to produce acute hepatic injury. Portal pressure, liver serum enzymes and ammonia plasma levels were determined. Brain cortex water content was registered and trypan blue was utilized to study blood brain barrier integrity. Reflexes and behavioral tests were recorded. RESULTS: Portal hypertension was significantly elevated in groups II and IV. Liver enzymes and ammonia plasma levels were increased in groups II, III and IV. Prehepatic portal hypertension (group II), acetaminophen intoxication (group III) and both (group IV) had changes in the blood brain-barrier integrity (trypan blue) and hyperammonemia. Cortical edema was present in rats with acute hepatic injury in groups III and IV. Behavioral test (rota rod) was altered in group IV. CONCLUSION: These results suggest the possibility of another pathway for cortical edema production because blood brain barrier was altered (vasogenic) and hyperammonemia was registered (cytotoxic). Group IV, with behavioral altered test, can be considered as a model for study at an early stage of portal-systemic encephalopathy.

Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system.

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.

Effect of neurogenic stress and ethanol on nitric oxide synthase and cyclooxygenase activities in rat adrenals.

Repeated restraint stress (RRS) in male rats activated the pituitary adrenal system, as indicated by increases in adrenal weight and plasma corticosterone concentration that were accompanied by a decrease in constitutive nitric oxide synthase (cNOS), but not inducible NOS (iNOS). iNOS activated cyclooxgenase, causing elevated prostaglandin E(2) (PGE(2)) and F(2 alpha) in the adrenals, but had no effect on lipoxygenase. Administration of ethanol (ETOH) was also associated with elevated adrenal weight and a slight increase in corticosterone coupled with a decrease in both cNOS and iNOS and PGs in the adrenal. When ETOH was administered together with RRS, a decrease in iNOS and PGE release was noted consequent to a reduction in iNOS. Thus, ETOH probably reduced RRS-induced adrenocorticotropic hormone release. Adrenals were incubated in vitro to further evaluate the role of NO in these processes. Results indicated that NO released by sodium nitroprusside increased corticosterone release presumably by activating guanylyl cyclase with production of cyclic guanosine monophosphate (cGMP), because although NO also increased PGE release, PGE(2) (10(-5)-10(-9) M) decreased corticosterone release, an effect that was highly significant at a concentration of 10(-7) M PGE(2). ETOH (100 mM) had no effect on corticosterone release and did not block the increase in corticosterone caused by NO; however, ETOH reduced PGE release into the medium and blocked PGE(2) release induced by NO. Consequently, NO activated corticosterone release not by PGs, but by activation of guanylyl cyclase and release of cGMP. PGs have a negative feedback to suppress corticosterone release.