The serotonergic system may be involved in the sleep-inducing action of oleamide in rats

The mechanism underlying the sleep-inducing effect of oleamide, an endogenous fatty acid amide, was studied in rats. Animals implanted with cerebrocortical and dorsal neck muscle electrodes were monitored continuously by electroencephalograph (EEG) and electromyograph (EMG) for 4 h after i.p. or s.c. injection of drugs. Oleamide induced a dose-dependent increase in slow-wave sleep (SWS), a decrease in wakefulness (W) and sleep latency, but had no effect on rapid-eye-movement sleep (REMS). The oleamide-induced increase in SWS was prevented by 5-HT reuptake inhibitors such as fluoxetine or fenfluramine and by agonists at 5-HT_1A receptors such as buspirone or 8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT). Moreover, the selective 5-HT_1A receptor antagonist WAY100635 markedly antagonized the suppression of the oleamide-induced increase in SWS by 8-OH-DPAT. These data provide the first behavioural evidence that the serotonergic system may be involved in the sleep-inducing action of oleamide in rats.     

Acute and subchronic administration of anandamide or oleamide increases REM sleep in rats

Anandamide and oleamide, induce sleep when administered acutely, via the CB1 receptor. Their subchronic administration must be tested to demonstrate the absence of tolerance to this effect, and that the sudden withdrawal of these endocannabinoids (eCBs) does not affect sleep negatively. The sleep-waking cycle of rats was evaluated for 24 h, under the effect of an acute or subchronic administration of eCBs, and during sudden eCBs withdrawal. AM251, a CB1 receptor antagonist (CB1Ra) was utilized to block eCBs effects. Our results indicated that both acute and subchronic administration of eCBs increase REMS. During eCBs withdrawal, rats lack the expression of an abstinence-like syndrome. AM251 was efficacious to prevent REMS increase caused by both acute and subchronic administration of these eCBs, suggesting that this effect is mediated by the CB1 receptor. Our data further support a role of the eCBs in REMS regulation.     

缝隙连接增强肿瘤坏死因子-α的肝细胞毒性

【目的】 观察缝隙连接(GJ)功能改变对肿瘤坏死因子-α(TNF-α)肝细胞毒性的影响?【方法】 使用3种不同的方法:(1)GJ抑制剂预处理;(2)高低密度接种细胞;(3)小分子RNA转染抑制缝隙连接蛋白32(Cx32)表达等3种不同的方法抑制GJ的功能,观察不同浓度TNF-α作用不同时间后对肝细胞毒性的影响?【结果】 TNF-α的肝细胞毒性呈浓度和时间依赖性;抑制剂预处理后,TNF-α的肝细胞毒性明显降低(P < 0.01);低密度接种细胞(细胞间无GJ形成)时,TNF-α的肝细胞毒性较高密度接种细胞(细胞间有GJ形成)明显降低(P < 0.01);抑制Cx32的表达后,TNF-α的肝细胞毒性明显降低(P < 0.01)? 【结论】 抑制由Cx32组成的GJ的功能后,TNF-α的肝细胞毒性明显降低,Cx32的表达对于TNF-α的肝细胞毒性作用具有重要的作用?     

Enhanced radiosensitization of p53 mutant cells by oleamide

PURPOSE: Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. METHODS AND MATERIALS: NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. RESULTS: Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. CONCLUSIONS: Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.     

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation.     

Fatty acid amide hydrolase in brain ventricular epithelium: mutually exclusive patterns of expression in mouse and rat

Fatty acid amides and fatty acid ethanolamides are novel signalling molecules exemplified by the sleep-inducing lipid oleamide and the endocannabinoid anandamide, respectively. These substances are inactivated by fatty acid amide hydrolase (FAAH), an enzyme that is expressed by neurons and non-neuronal cells in the brain. In the rat, FAAH-immunoreactivity has been detected in epithelial cells of the choroid plexus and, in accordance with this finding, here we report FAAH mRNA expression in rat choroid plexus epithelium using in situ hybridisation methods. Surprisingly, a comparative analysis of mouse brain did not reveal FAAH mRNA expression or FAAH-immunoreactivity in the choroid plexus of this species. FAAH-immunoreactivity was, however, detected in non-choroidal ventricular ependymal cells in the mouse brain and the specificity of this immunostaining was confirmed by analysis of FAAH-knockout mice. FAAH-immunoreactivity was detected in ependymal cells throughout the ventricles of the mouse brain but with regional variation in the intensity of immunostaining. Intriguingly, in rat brain, although FAAH expression is observed in choroid plexus epithelial cells, little or no FAAH-immunoreactivity is present in the ventricular ependyma. Thus, there are mutually exclusive patterns of FAAH expression in the ventricular epithelium of rat and mouse brain. Our observations provide the basis for an experimental analysis that exploits differences in FAAH expression in rat and mouse to investigate FAAH function in ventricular epithelial cells and, in particular, the role of FAAH in regulating the sleep-inducing agent oleamide in cerebrospinal fluid.     

Oleamide: A Fatty Acid Amide Signaling Molecule in the Cardiovascular System?

Oleamide (cis‐9,10‐octadecenoamide), a fatty acid primary amide discovered in the cerebrospinal fluid of sleep‐deprived cats, has a variety of actions that give it potential as a signaling molecule, although these actions have not been extensively investigated in the cardiovascular system. The synthetic pathway probably involves synthesis of oleoylglycine and then conversion to oleamide by peptidylglycine α?amidating monooxygenase (PAM); breakdown of oleamide is by fatty acid amide hydrolase (FAAH). Oleamide interacts with voltage‐gated Na⁺ channels and allosterically with GABA_A and 5‐HT₇ receptors as well as having cannabinoid‐like actions. The latter have been suggested to be due to potentiation of the effects of endocannabinoids such as anandamide by inhibiting FAAH‐mediated hydrolysis. This might underlie an “entourage effect” whereby co‐released endogenous nonagonist congeners of endocannabinoids protect the active molecule from hydrolysis by FAAH. However, oleamide has direct agonist actions at CB₁ cannabinoid receptors and also activates the TRPV1 vanilloid receptor. Other actions include inhibition of gap‐junctional communication, and this might give oleamide a role in myocardial development. Many of these actions are absent from the trans isomer of 9,10‐octadecenoamide. One of the most potent actions of oleamide is vasodilation. In rat small mesenteric artery the response does not involve CB₁ cannabinoid receptors but another pertussis toxin‐sensitive, G protein–coupled receptor, as yet unidentified. This receptor is sensitive to rimonabant and O‐1918, an antagonist at the putative “abnormal‐cannabidiol” or endothelial “anandamide” receptors. Vasodilation is mediated by endothelium‐derived nitric oxide, endothelium‐dependent hyperpolarization, and also through activation of TRPV1 receptors. A physiological role for oleamide in the heart and circulation has yet to be demonstrated, as has production by cells of the cardiovascular system, but this molecule has a range of actions that could give it considerable modulatory power.     

The sleep lipid oleamide may represent an endogenous anticonvulsant: an in vitro comparative study in the 4-aminopyridine rat brain-slice model

cis-Oleamide (cOA) is a putative endocannabinoid, which modulates GABA(A) receptors, Na+ channels and gap-junctions (important targets for clinical and experimental anticonvulsants). Here we address the hypothesis that cOA possesses seizure limiting properties and might represent an endogenous anticonvulsant. Field potentials were recorded from the rat hippocampus and visual cortex. The effects of cOA, were compared to carbamazepine (CBZ), pentobarbital (PB) and carbenoxolone (CRX) on 4-Aminopyridine(4AP)-induced epileptiform discharges. CBZ (100 microM), PB (50 microM) and CRX (100 microM), but not cOA (64 microM), significantly attenuated the duration of the evoked epileptiform discharges in CA1. Interictal activity in CA3 was significantly depressed by CRX and cOA (irreversible by AM251), increased by CBZ and remained unaffected by PB. CBZ, PB and CRX abolished spontaneous ictal events and attenuated evoked ictal discharges in the visual cortex. cOA did not abolish spontaneous ictal events, but significantly (albeit weakly) reduced the duration of evoked ictal events. cOA and CRX, in contrast to CBZ or PB, caused a significant delay in the development of the evoked (tonic phase) epileptiform discharges. The weak effects of cOA seem independent of cannabinoid (CB1) receptors. Enzymatic cleavage and lack of specific antagonists for cOA confound simple interpretations of its actions in slices. Its high lipophilicity, imposing a permeability barrier, may also explain the lack of anticonvulsant activity. The effects of cOA may well be masked by release of the endogenous ligand upon ictal depolarisation as we demonstrate here for established endocannabinoids. cOA does not possess profound antiepileptic actions in our hands compared to CBZ, PB or CRX.     

Pharmacological enhancement of cannabinoid CB1 receptor activity elicits an antidepressant-like response in the rat forced swim test

These experiments aimed to assess whether enhanced activity at the cannabinoid CB₁ receptor elicits antidepressant-like effects. To examine this we administered 1 and 5 mg/kg doses of the endocannabinoid uptake inhibitor AM404; 5 and 25 μg/kg doses of HU-210, a potent CB₁ receptor agonist; 1, 2.5 and 5 mg/kg of oleamide, which elicits cannabinoidergic actions; 1 and 5 mg/kg doses of AM 251, a selective CB₁ receptor antagonist, as well as 10 mg/kg desipramine (a positive antidepressant control) and measured the duration of immobility, during a 5-min test session of the rat Porsolt forced swim test. Results demonstrated that administration of desipramine reduced immobility duration by about 50% and that all of AM404, oleamide and HU-210 administration induced comparable decreases in immobility that were blocked by pretreatment with AM 251. Administration of the antagonist AM 251 alone had no effect on immobility at either dose. These data suggest that enhancement of CB₁ receptor signaling results in antidepressant effects in the forced swim test similar to that seen following conventional antidepressant administration.     

Oleamide suppresses lipopolysaccharide-induced expression of iNOS and COX-2 through inhibition of NF-κB activation in BV2 murine microglial cells

Oleamide (cis-9-octadecenamide) is an endogenous sleep-inducing fatty acid amide that accumulates in the cerebrospinal fluid of the sleep-deprived animals. Microglia are the major immune cells involved in neuroinflammation causing brain damage during infection, ischemia, and neurodegenerative disease. In this study, we examined the effects of oleamide on LPS-induced production of proinflammatory mediators and the mechanisms involved in BV2 microglia. Oleamide inhibited LPS-induced production of NO and prostaglandin E2 as well as expression of iNOS and COX-2. We showed that oleamide blocked LPS-induced NF-κB activation and phosphorylation of inhibitor κB kinase (IKK). We also showed that oleamide inhibited LPS-induced phosphorylation of Akt, p38 MAPK, and ERK, activation of PI 3-kinase, and accumulation of reactive oxygen species (ROS). Finally, we showed that a specific antagonist of the CB2 receptor, AM630, blocked the inhibitory effects of oleamide on LPS-induced production of proinflammatory mediators and activation of NF-κB. Taken together, our results suggest that oleamide shows an anti-inflammatory effect through inhibition of NF-κB activation in LPS-stimulated BV2 microglia.