Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus

Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by approximately 2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This "FAAH activator" was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A(2), C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B(4) or prostaglandins E(2) and F(2)alpha had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated.     

Science signals a new understanding of marihuana

Some recent scientific advances in the study of the cannabinoids are outlined. The mode of action of marihuana and the cannabinoids has now been described. They belong to a new class of drug that acts on a hitherto undescribed neuro-physiological system. An endogenous neurotransmitter or neuromodulator for this system has been isolated, identified and named “anandamide”. These findings throw new light and imbue new confidence for the future of the therapeutic application of compounds derived from and related to the cannabinoids and anandamide. An outline is also provided of the current knowledge and future potential of cannabinoids in therapeutics. The effect of the current legal classification of the cannabinoids on the research and development of these compounds is discussed.     

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System

Background:

Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses.

Methods:

We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated.

Results:

Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning.

Conclusion:

These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased.     

Enhanced vasorelaxation effect of endogenous anandamide on thoracic aorta in renal vascular hypertension rats

Emerging evidence has indicated that anandamide (AEA) is able to stimulate vasorelaxation in both spontaneously hypertensive rats (SHRs) and L‐NAME‐induced hypertensive rats. Yet it remains unknown whether AEA modulates vasomotion of the aorta in renovascular hypertensive (RVH) rats. The aim of present study is to explore the effect of AEA on the relaxation of thoracic aortas in two‐kidney one‐clip (2K1C)‐induced RVH rats. It is demonstrated that AEA stimulates a pronounced relaxation in the aortas of 2K1C rats compared with sham rats. The enhanced relaxation caused by AEA in aortas from 2K1C rats was diminished in the presence of the cannabinoid receptor‐1 (CB₁) antagonist AM251 and the CB₂ receptor antagonist AM630. Likewise, the vasodilation action of AEA was blocked in L‐NAME‐treated or endothelium‐denuded aortas. The Western blot results revealed that the expression of CB₁ and CB₂ receptors was increased in the 2K1C rat aortas compared with sham rats. The phosphorylation of endothelial nitric oxide synthase (p‐eNOS) at the activation site Ser1177 was enhanced in AEA‐treated rings from 2K1C rats in both time‐dependent and dose‐dependent manners. The augmented p‐eNOS expression was inhibited by the co‐treatment with AM251 or AM630. Taken together, the present study demonstrated that AEA enhanced endothelium‐dependent aortic relaxation through activation of both CB₁ and CB₂ receptors and P‐eNOS/NO pathway in 2K1C rats.     

Temporal changes in N‐acylethanolamine content and metabolism throughout the peri‐adolescent period

Fatty acid amide hydrolase (FAAH) regulates tissue concentrations of N‐acylethanolamines (NAEs), including the endocannabinoid, N‐arachidonylethanolamide (anandamide, AEA). FAAH activity and NAEs are widely distributed throughout the brain and FAAH activity regulates an array of processes including emotion, cognition, inflammation, and feeding. However, there is relatively little research describing how this system develops throughout adolescence, particularly within limbic circuits regulating stress and reward processing. Thus, this study characterized temporal changes in NAE content (AEA, oleoylethanolamine [OEA], and palmitoylethanolamide [PEA]) and FAAH activity across the peri‐adolescent period, in four corticolimbic structures (amygdala, hippocampus, prefrontal cortex, and hypothalamus). Brain tissue of male Sprague–Dawley rats was collected on postnatal days (PND) 25, 35, 45, and 70, representing pre‐adolescence, early‐ to mid‐adolescence, late adolescence, and adulthood, respectively. Tissue was analyzed for AEA, OEA, and PEA content as well as FAAH activity at each time point. AEA, OEA, and PEA exhibited a similar temporal pattern in all four brain regions. NAE concentrations were lowest at PND 25 and highest at PND 35. NAE concentrations decreased between PNDs 35 and 45 and increased between PNDs 45 and 70. FAAH activity mirrored the pattern of NAE content in which it decreased between PNDs 25 and 35, increased between PNDs 35 and 45, and decreased between PNDs 45 and 70. These age‐dependent patterns of NAE content and FAAH activity demonstrate temporal specificity to the development of this system and could contribute to alterations in stress sensitivity, emotionality, and executive function which also fluctuate during this developmental period. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

蛋白激酶C 参与内源性大麻素花生四烯乙醇胺对L型钙电流的抑制作用

目的：研究内源性大麻素物质花生四烯乙醇胺是否通过改变蛋白激酶C（ PKC）活性从而抑制心肌L型钙电流,并进一步探讨可能改变PKC活性的信号途径。方法应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流（P ＜0 n．05）;应用PepTag非放射性蛋白激酶C检测系统（ Promega）检测PKC活性;Elisa试剂盒测定细胞中二脂酰甘油（ DAG）的含量;western blot 技术测定磷脂酶Cβ（ PLCβ）和磷酸化磷脂酶C β（ p-PLCβ）表达。结果应用花生四烯乙醇胺灌流心肌细胞后显著抑制心肌L型钙电流（ P ＜0．05）,预先应用大麻素1型受体（ CB1）阻断剂AM251或PKC非特异性激动剂佛波醇酯（PMA）可以完全阻断此抑制效应,而大麻素2型受体（CB2）阻断剂AM630没有阻断花生四烯乙醇胺抑制L型钙电流的作用。检测心肌细胞PKC活性发现,花生四烯乙醇胺明显抑制PKC活性（ P ＜0．05）,同样预先应用AM251或PMA完全阻断花生四烯乙醇胺对PKC活性的抑制效应,而AM630无此效应。应用花生四烯乙醇胺没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论本实验首次证明内源性大麻素花生四烯乙醇胺激活心肌细胞CB1受体后抑制细胞PKC的活性,从而抑制L型钙电流,此过程没有PLCβ-DAG途径参与。     

Dynamic changes of anandamide in the cerebrospinal fluid of Parkinson's disease patients

A correct balance between endocannabinoid and dopamine‐dependent systems is believed to underlie physiological motor control. We measured the levels of the endocannabinoid anandamide in the cerebrospinal fluid of Parkinson's disease (PD) patients. Subjects were divided into three groups: newly diagnosed de novo patients, subjects undergoing drug withdrawal, and patients under pharmacological therapy. These groups were compared to age‐matched control subjects. Anandamide levels in untreated patients were more than doubled as compared to controls. However, chronic dopaminergic replacement restored control anandamide levels. Abnormal anandamide increase might reflect a compensatory mechanism occurring in course of PD, aimed at normalizing dopamine depletion. © 2010 Movement Disorder Society