Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.     

Heregulin upregulates the expression of nitric oxide synthase (NOS)-1 in rat cerebellar granule neurons via the ErbB4 receptor

Heregulin plays key roles in regulating cell number, determining fate and establishing pattern in the developing nervous system via specific receptors (ErbBs), including ErbB4. Two recent reports have shown that ErbB4 forms a complex with postsynaptic density proteins, which are, in turn, known to complex with nitric oxide synthase (NOS)-1. To reveal whether heregulin might regulate the expression of NOS-1, cultures enriched in cerebellar granule cells were exposed to heregulin for 72 h. This treatment resulted in an increase in NOS-1 protein (> 70%), an effect mediated by the ErbB4 receptor. While nitric oxide might mediate some of the downstream effects of heregulin in the nervous system, heregulin treatment neither enhanced granule cell survival, nor protected neurons from acute glutamate excitotoxicity.     

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Discovery of indole alkaloids with cannabinoid CB1 receptor antagonistic activity

Three indole alkaloids, voacamine (1), 3,6-oxidovoacangine (2), and a new alkaloid, 5-hydroxy-3,6-oxidovoacangine (3), isolated from Voacanga africana were found to exhibit potent cannabinoid CB1 receptor antagonistic activity. This is the first example of CB1 antagonists derived from natural alkaloids.     

Sequential activation of constitutive and inducible nitric oxide synthase (NOS) in rat cerebellar granule neurons by pseudomonas fluorescens and invasive behaviour of the bacteria

Previous studies have shown that Pseudomonas fluorescens and its lipopolysaccharide (LPS) exert dose-related cytotoxic effects on neurons and glial cells. In the present work, we investigated the time course effect of P. fluorescens MF37 and its LPS on cultured rat cerebellar granule neurons. The kinetics of binding of P. fluorescens to cerebellar granule neurons is rapid and reaches a mean of 3 bacteria/cell after 5 h. As demonstrated by measurement of the concentration of nitrite in the culture medium, P. fluorescens induces a rapid stimulation (3 h) of the nitric oxide synthase (NOS) activity of the cells. In contrast, LPS extracted from P. fluorescens requires a long lag phase (24 h) before observation of an activation of NOS. Measurement of the membrane resting potential of granule neurons showed that within 3 h of incubation there was no difference of effect between the action of P. fluorescens and that of its endotoxin. Two complementary approaches allowed to demonstrate that P. fluorescens MF37 presents a rapid invasive behaviour suggesting a mobilisation of calcium in its early steps of action. The present study reveals that P. fluorescens induces the sequential activation of a constitutive calcium-dependent NOS and that of an inducible NOS activated by LPS. Our results also suggest that in P. fluorescens cytotoxicity and invasion are not mutually exclusive events.     

Highly selective CB(1) cannabinoid receptor ligands and novel CB(1)/VR(1) vanilloid receptor "hybrid" ligands

Anandamide and the metabolically stabler analogs, (R)-1'-methyl-2'-hydroxy-ethyl-arachidonamide (Met-AEA) and N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide (arvanil), are CB(1) cannabinoid and VR(1) vanilloid receptors agonists. We synthesized 1',1'-dimethylheptyl-arvanil (O-1839) and six other AEA analogs obtained by addition of either a hydroxy, cyano, or bromo group on the C-20 atom of 1,1'-dimethylpentyl-Met-AEA (O-1811, O-1812 and O-1860, respectively) or 1,1'-dimethylpentyl-arvanil (O-1856, O-1895 and O-1861, respectively). The compounds were tested for their (i) affinity for CB(1) and CB(2) receptors, (ii) capability to activate VR1 receptors, (iii) inhibitory effect on the anandamide hydrolysis and on the anandamide membrane transporter, and (iv) cannabimimetic activity in the mouse 'tetrad' of in vivo assays. O-1812 is the first ligand ever proven to be highly (500- to 1000-fold) selective for CB(1) vs both VR(1) and CB(2) receptors, while O-1861 is the first true "hybrid" agonist of CB(1)/VR(1) receptors and a compound with potential therapeutic importance. The activities of the seven compounds in vivo did not correlate with their activities at either CB(1) or VR(1) receptors, thus suggesting the existence of other brain sites of action mediating some of their neurobehavioral actions in mice.     

Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation

The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5mg/kg) robustly increased dopamine and norepinephrine release (to ～175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ～135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.     

Ligand based structural studies of the CB1 cannabinoid receptor.

The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.     

Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis

Using a well documented ex vivo system consisting of rodent cerebellar granule cells (CGCs) the activation of caspases 3 and 6 during apoptosis induced by withdrawal of trophic support was analyzed. At the time of deprivation, the addition of the irreversible, broad-spectrum caspase inhibitor zVADfmk or the cell permeable, caspase 6 inhibitor CP-VEID-cho can transiently suppress the appearance of apoptosis, including the early appearance of DNA fragmentation. Using immunoblotting and fluorogenic peptide assays we observe deprivation-induced activation of caspases 3 and 6, but not caspase 9. Furthermore, active caspase 6 is capable of processing and activating procaspase 3 in cellular extracts prepared from non-apoptotic CGCs, whereas caspase 3 failed to activate caspase 6. In consonant with this, the cell permeable caspase 6 inhibitor prevented deprivation-induced caspase 3 activation whereas a cell permeable caspase 3 inhibitor, CP-DEVD-cho, had no effect on caspase 6 activation. This would indicate that caspase 6 is a significant inducer of the early caspase 3 activity in apoptotic CGCs.     

Rapid activation of endothelial nitric oxide synthase by estrogen.

Estrogen is an important atheroprotective molecule that causes the rapid dilation of blood vessels by stimulating endothelial nitric oxide synthase (eNOS). There is also evidence that estrogen modulates airway epithelial NO production, thereby potentially affecting bronchial hyperresponsiveness. Studies in cultured endothelial and airway epithelial cells indicate that physiologic concentrations of estrogen cause rapid direct activation of eNOS that is unaffected by actinomycin D, but fully inhibited by estrogen receptor (ER) antagonism. Overexpression of ERalpha leads to marked enhancement of the acute response to estrogen, and this process is blocked by ER antagonism, it is specific to estrogen, and it requires the ERalpha hormone binding domain. In addition, the acute response of eNOS to estrogen can be reconstituted in COS-7 cells cotransfected with wild-type ERalpha and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of calcium influx, or tyrosine kinases or MAP kinase prevents the stimulation of eNOS by estrogen, and estrogen causes rapid ER-dependent activation of MAP kinase. These findings indicate that the acute effects of estrogen on both endothelial and airway epithelial eNOS are mediated by ERalpha functioning in a novel, nongenomic manner to activate the enzyme via calcium-dependent, MAP kinase-dependent mechanisms.     

Vascular and perivascular nitric oxide release and transport: biochemical pathways of neuronal nitric oxide synthase (NOS1) and endothelial nitric oxide synthase (NOS3)

Nitric oxide (NO) derived from nitric oxide synthase (NOS) is an important paracrine effector that maintains vascular tone. The release of NO mediated by NOS isozymes under various O(2) conditions critically determines the NO bioavailability in tissues. Because of experimental difficulties, there has been no direct information on how enzymatic NO production and distribution change around arterioles under various oxygen conditions. In this study, we used computational models based on the analysis of biochemical pathways of enzymatic NO synthesis and the availability of NOS isozymes to quantify the NO production by neuronal NOS (NOS1) and endothelial NOS (NOS3). We compared the catalytic activities of NOS1 and NOS3 and their sensitivities to the concentration of substrate O(2). Based on the NO release rates predicted from kinetic models, the geometric distribution of NO sources, and mass balance analysis, we predicted the NO concentration profiles around an arteriole under various O(2) conditions. The results indicated that NOS1-catalyzed NO production was significantly more sensitive to ambient O(2) concentration than that catalyzed by NOS3. Also, the high sensitivity of NOS1 catalytic activity to O(2) was associated with significantly reduced NO production and therefore NO concentrations, upon hypoxia. Moreover, the major source determining the distribution of NO was NOS1, which was abundantly expressed in the nerve fibers and mast cells close to arterioles, rather than NOS3, which was expressed in the endothelium. Finally, the perivascular NO concentration predicted by the models under conditions of normoxia was paradoxically at least an order of magnitude lower than a number of experimental measurements, suggesting a higher abundance of NOS1 or NOS3 and/or the existence of other enzymatic or nonenzymatic sources of NO in the microvasculature.     

Constitutive endocytic cycle of the CB1 cannabinoid receptor

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.     

Loss of cannabinoid receptor CB1 induces preterm birth

Background

Preterm birth accounting approximate 10% of pregnancies in women is a tremendous social, clinical and economic burden. However, its underlying causes remain largely unknown. Emerging evidence suggests that endocannabinoid signaling via cannabinoid receptor CB1 play critical roles in multiple early pregnancy events in both animals and humans. Since our previous studies demonstrated that loss of CB1 defers the normal implantation window in mice, we surmised that CB1 deficiency would influence parturition events.

Methods and Findings

Exploiting mouse models with targeted deletion of Cnr1, Cnr2 and Ptgs1 encoding CB1, CB2 and cyclooxygenase-1, respectively, we examined consequences of CB1 or CB2 silencing on the onset of parturition. We observed that genetic or pharmacological inactivation of CB1, but not CB2, induced preterm labor in mice. Radioimmunoassay analysis of circulating levels of ovarian steroid hormones revealed that premature birth resulting from CB1 inactivation is correlated with altered progesterone/estrogen ratios prior to parturition. More strikingly, the phenotypic defects of prolonged pregnancy length and parturition failure in mice missing Ptgs1 were corrected by introducing CB1 deficiency into Ptgs1 null mice. In addition, loss of CB1 resulted in aberrant secretions of corticotrophin-releasing hormone and corticosterone during late gestation. The pathophysiological significance of this altered corticotrophin-releasing hormone-driven endocrine activity in the absence of CB1 was evident from our subsequent findings that a selective corticotrophin-releasing hormone antagonist was able to restore the normal parturition timing in Cnr1 deficient mice. In contrast, wild-type females receiving excessive levels of corticosterone induced preterm birth.

Conclusions

CB1 deficiency altering normal progesterone and estrogen levels induces preterm birth in mice. This defect is independent of prostaglandins produced by cyclooxygenase-1. Moreover, CB1 inactivation resulted in aberrant corticotrophin-releasing hormone and corticosterone activities prior to parturition, suggesting that CB1 regulates labor by interacting with the corticotrophin-releasing hormone-driven endocrine axis.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.