Quantification of anandamide content in animal cells and tissues: the normalization makes the difference

Anandamide (N-arachidonoylethanolamine, AEA) is an endogenous lipid that binds to cannabinoid receptors in the central nervous system and in peripheral cells. Quantitative analysis of AEA is generally based on the normalization to the fresh weight of the samples. Here, we show that the normalization procedure of AEA content is such a critical factor, that it might introduce per se significant discrepancies in the quantification of AEA even in the same sample. We suggest that a rapid, accurate and most reliable reference to quantify AEA and congeners from different sources is the protein content, a common parameter to cells and tissues

Anandamide uptake by synaptosomes from human, mouse and rat brain: inhibition by glutamine and glutamate

Anandamide (N-arachidonoylethanolamine, AEA) belongs to an emerging class of endogenous lipids, called "endocannabinoids". A specific AEA membrane transporter (AMT) allows the import of this lipid and its degradation by the intracellular enzyme AEA hydrolase. Here, we show that synaptosomes from human, mouse and rat brain might be an ideal ex vivo system for the study of: i) the accumulation of AEA in brain, and ii) the pharmacological properties of AMT inhibitors. Using this ex vivo system, we demonstrate for the first time that glutamine and glutamate act as non-competitive inhibitors of AEA uptake by human, mouse and rat brain AMT

Leptin activates the anandamide hydrolase promoter in human T lymphocytes through STAT3

Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility

Anandamide induces apoptosis in human cells via vanilloid receptors. Evidence for a protective role of cannabinoid receptors.

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitor Nomega-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors

Progesterone activates fatty acid amide hydrolase (FAAH) promoter in human T lymphocytes through the transcription factor Ikaros. Evidence for a synergistic effect of leptin.

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human T lymphocytes, up to a ∼270% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level and was specific. Indeed, neither the activity of the anandamide-synthesizing N-acyltransferase and phospholipase D, nor the activity of the anandamide transporter, nor the binding to cannabinoid receptors were affected by progesterone under the same experimental conditions. The activation of FAAH by progesterone was paralleled by a decrease (down to 60%) of the cellular levels of anandamide and involved increased nuclear levels of the transcription factor Ikaros. Analysis of the FAAH promoter showed an Ikaros binding site, and mutation of this site prevented FAAH activation by progesterone in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Furthermore, the effect of progesterone on FAAH promoter was additive to that of physiological amounts of leptin, which binds to a cAMP response element-like site in the promoter region. Taken together, these results suggest that progesterone and leptin, by up-regulating the FAAH promoter at different sites, enhance FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility

Kinetics of Electron Transfer between Azurin and Cytochrome 551 from Pseudomonas

Abstract The kinetics of electron transfer between the copper-containing protein azurin (Cu++/Cu+) and cytochrome 551 (Fe+++/Fe++) from Pseudomonas has been studied by rapid mixing methods. The reaction in both directions is fast; at low reagent concentrations (∼10-6 m) the apparent second order rate constant, at 20°, is about 3 x 106 m-1 sec-1 for the reaction Fe++ + Cu++ and 1.4 x 106 m-1 sec-1 for the reaction Fe+++ + Cu+. At high reagent concentrations the rates tend to reach a limiting value indicating that the reaction is not a simple second order process. The kinetics of the reactions of the reduced and oxidized forms of azurin and Pseudomonas cytochrome 551 with ferricyanide and dithionite has also been investigated. The rates of these reactions, at comparable reagent concentrations, are orders of magnitude lower than that between azurin and cytochrome 551

Anandamide Uptake by Human Endothelial Cells and Its Regulation by Nitric Oxide

Anandamide (AEA) has vasodilator activity, which can be terminated by cellular re-uptake and degradation. Here we investigated the presence and regulation of the AEA transporter in human umbelical vein endothelial cells (HUVECs). HUVECs take up AEA by facilitated transport (apparent K(m) = 190 +/- 10 nm and V(max) = 45 +/- 3 pmol. min(-1).mg(-1) protein), which is inhibited by alpha-linolenoyl-vanillyl-amide and N-(4-hydroxyphenyl)-arachidonoylamide, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scavenger hydroxocobalamin abolishes the latter effect, which is instead enhanced by superoxide anions but inhibited by superoxide dismutase and N-acetylcysteine, a precursor of glutathione synthesis. Peroxynitrite (ONOO(-)) causes a 4-fold activation of AEA transport into cells. The HUVEC AEA transporter contributes to the termination of a typical type 1 cannabinoid receptor (CB(1)) -mediated action of AEA, i.e. the inhibition of forskolin-stimulated adenylyl cyclase, because NO/ONOO(-) donors and alpha-linolenoyl-vanillyl-amide/N-(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, respectively, this effect of AEA. Consistently, activation of CB(1) cannabinoid receptors by either AEA or the cannabinoid HU-210 caused a stimulation of HUVEC inducible NO synthase activity and expression up to 2.9- and 2. 6-fold, respectively. Also these effects are regulated by the AEA transporter. HU-210 enhanced AEA uptake by HUVECs in a fashion sensitive to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester. These findings suggest a NO-mediated regulatory loop between CB(1) cannabinoid receptors and AEA transporter

The Activity of Anandamide at Vanilloid VR1 Receptors Requires Facilitated Transport across the Cell Membrane and Is Limited by Intracellular Metabolism

The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhanced anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A inhibited also the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism

Urinary physiology and hypoxia: a pilot study of moderate-altitude trekking effects on urodynamic indexes

Exposure to high altitude is one of the most widely used models to study the adaptive response to hypoxia in humans. However, little is known about the related effects on micturition. The present study addresses the adaptive urinary responses in four healthy adult lowlanders, comparing urodynamic indexes at Kathmandu [1,450 m above sea level (a.s.l.); K1450] and during a sojourn in Namche Bazar (3,500 m a.s.l.; NB3500). The urodynamic testing consisted of cistomanometry and bladder pressure/flow measurements. Anthropometrics, electrocardiographic, and peripheral capillary oxygen saturation data were also collected. The main findings consisted of significant reductions in bladder power at maximum urine flow by ~30%, bladder contractility index by 13%, and infused volume both at first (by 57%) and urgency sensation (by 14%) to urinate, indicating a reduced cystometric capacity, at NB3500. In addition to the urinary changes, we found that oxygen saturation, body mass index, body surface area, and median RR time were all significantly reduced at altitude. We submit that the hypoxia-related parasympathetic inhibition could be the underlying mechanism of both urodynamic and heart rate adaptive responses to high-altitude exposure. Moreover, increased diuresis and faster bladder filling at altitude may trigger the anticipation of being able to void, a common cause of urgency. We believe that the present pilot study represents an original approach to the study of urinary physiology at altitude

T1 Bladder Cancer: Comparison of the Prognostic Impact of Two Substaging Systems on Disease Recurrence and Progression and Suggestion of a Novel Nomogram

Background: The T1 substaging of bladder cancer (BCa) potentially impacts disease progression. The objective of the study was to compare the prognostic accuracy of two substaging systems on the recurrence and progression of primary pathologic T1 (pT1) BCa and to test a nomogram based on pT1 substaging for predicting recurrence-free survival (RFS) and progression-free survival (PFS).Methods: The medical records of 204 patients affected by pT1 BCa were retrospectively reviewed. Substaging was defined according to the depth of lamina propria invasion in T1(a-c) and the extension of the lamina propria invasion to T1-microinvasive (T1(m)) or T1-extensive (T1(e)). Uni- and multivariable Cox regression models evaluated the independent variables correlated with recurrence and progression. The predictive accuracies of the two substaging systems were compared by Harrell's C index. Multivariate Cox regression models for the RFS and PFS were also depicted by a nomogram.Results: The 5-year RFS was 47.5% with a significant difference between T1(c) and T1(a) (p = 0.02) and between T1(e) and T1(m) (p < 0.001). The 5-year PFS was 75.9% with a significant difference between T1(c) and T1(a) (p = 0.011) and between T1(e) and T1(m) (p < 0.001). Model T1(m-e) showed a higher predictive power than T1(a-c) for predicting RFS and PFS. In the univariate and multivariate model subcategory T1e, the diameter, location, and number of tumors were confirmed as factors influencing recurrence and progression after adjusting for the other variables. The nomogram incorporating the T1(m-e) model showed a satisfactory agreement between model predictions at 5 years and actual observations.Conclusions: Substaging is significantly associated with RFS and PFS for patients affected by T1 BCa and should be included in innovative prognostic nomograms