New tetrazole-based selective anandamide uptake inhibitors

A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC₅₀ = 2.3–5.1 μM) and selectively over other proteins involved in endocannabinoid action and metabolism.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase

Fatty acid amide hydrolase (EC 3.5.1.4.) is the enzyme responsible for the rapid degradation of lipid-derived chemical messengers such as anandamide, oleamide, and 2-arachidonoylglycerol. The pharmacological characterization of this enzyme in vivo has been hampered by the lack of selective and bioavailable inhibitors. We have developed a simple, radioactive, high-throughput-compatible assay for this enzyme based on the differential absorption of the substrate and its products to activated charcoal. The assay was validated using known inhibitors. It may be applied for the identification of new inhibitors from a compound library.     

Simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonylglycerol) and isoprostane (8-epiprostaglandin F2alpha) by gas chromatography-mass spectrometry-selected ion monitoring for medical samples

This article describes the overall procedure for the simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonyglycerol) and isoprostane by gas chromatography-mass spectrometry in the selected-ion monitoring SIM mode (GC-MS-SIM) for medical samples. It also describes the general points of this method which a scientist who wants to assay a new, unidentified prostanoids and related compounds in medical samples would need to be clarified. The similar structures of prostaglandins, thromboxane, their metabolites, isoprostane, and arachidonyl compounds, allow them to be assayed after the simultaneous preparation of a single sample. The dimethyl isopropylsilyl ether forms of derivatized compounds are suitable for multiple GC-MS-SIM assay because of their molecular stability, and because they produce positive, strong, and large fragments on MS.     

Evolutionary conservation of physical and functional interactions between phospholipase D and actin

Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown. Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs. Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin. The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM. The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity. Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1. Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution.     

Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.     

Dual modulation of CNS voltage-gated calcium channels by cannabinoids: Focus on CB1 receptor-independent effects

The neuromodulatory effects of cannabinoids in the central nervous system have mainly been associated with G-protein coupled cannabinoid receptor (CB1R) mediated inhibition of voltage-gated calcium channels (VGCCs). Numerous studies show, however, that cannabinoids can also modulate VGCCs independent of CB1R activation. Nevertheless, despite the fact that endocannabinoids have a nearly equal efficacy for direct and CB1R-mediated effects on VGCC, the role of the direct cannabinoid–VGCC interaction has been largely underestimated.In this review, we summarize recent studies on the modulation of different types of VGCCs by cannabinoids, highlight the evidence for and implications of the CB1R-independent modulation, and put forward the concept, that direct interaction of cannabinoids and VGCCs is as important in regulation of VGCCs function as the CB1R-mediated effects.     

The transphosphatidylation activity of phospholipase D

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.     

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.     

Modulation of phospholipase D activity in vitro

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP₂). In a sophisticated interplay PIP₂ seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca²⁺ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed.     

Synthesis of phosphatidylated-monoterpene alcohols catalyzed by phospholipase D and their antiproliferative effects on human cancer cells

In order to prepare functional phospholipids in the medical and pharmaceutical fields, perillyl alcohol, myrtenol, and nerol were transphosphatidylated via phospholipase D in an aqueous system. The yields of phosphatidyl-perillyl alcohol, -myrtenol, and -nerol were 79 mol %, 87 mol %, and 91 mol %, respectively. The synthetic phosphatidylated monoterpenes showed a markedly antiproliferative effect on human prostate PC-3 and human leukemia HL-60 cells at 100 μM, while the free monoterpene alcohols had no effect at 400 μM.     

Anandamide-derived Prostamide F2α Negatively Regulates Adipogenesis

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB₁ receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F_2α ethanolamide (PGF_2αEA), of which bimatoprost is a potent synthetic analog. PGF_2αEA/bimatoprost act via prostaglandin F_2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF_2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF_2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF_2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF_2αEA versus prostaglandin F_2α biosynthesis accelerates adipogenesis. PGF_2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.     

Phosphatidylcholine-specific phospholipase C-mediated induction of phospholipase D activity in Fas-expressing murine cells

We have previously reported that Fas cross-linking resulted in the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and the subsequent activation of protein kinase C (PKC) and phospholipase D (PLD) in A20 cells. In an attempt to correlate the existence of PC-PLC activity and activation of PLD by Fas activation among various Fas-expressing murine cell lines, we have investigated the effect of anti-Fas monoclonal antibody on PC-PLC and PLD activities in A20, P388D1 and YAC-1 cell lines. Upon treatment of anti-Fas monoclonal antibody to these three cell lines, the activation of PLD was only observed in A20 cells. When the effect of anti-Fas monoclonal antibody on PKC and PC-PLC activities in Fas-expressing clones were investigated, the activation of PKC and PC-PLC was detected only in A20 clones. Results presented here also show that exogenous addition of Bacillus cereus PC-PLC activates PC hydrolysis, PKC and PLD in all three murine cell lines. These findings suggest that the activation of PC-PLC is a necessary requirement for the activation of PLD by Fas cross-linking and cell lines devoid of functional PC-PLC activity could exhibit enhanced PLD activity by exogenous addition of PC-PLC.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.