Dale Deutsch - Academia.edu (original) (raw)

Papers by Dale Deutsch

Springer eBooks, 2008

Only a few studies have addressed the transport of 2-arachidonoylglycerol (2-AG), a naturally occ... more Only a few studies have addressed the transport of 2-arachidonoylglycerol (2-AG), a naturally occurring agonist for cannabinoid receptors. Based upon saturation kinetics, these early reports have proposed that 2-AG enters the cell by a specific 2-AG transporter, via the putative anandamide transporter, or by simple diffusion. In this review, the uptake of 2-AG is discussed in light of the recent advances that have been made for anandamide transport, where the mechanism appears to be rate-limited diffusion through the membrane. Endocannabinoids may be a distinct class of agonists since they are hydrophobic and neutral, exhibiting similar biophysical properties to some anesthetics that freely diffuse through the membrane.

Life Sciences, Aug 1, 2005

Anandamide (AEA) uptake has been described over the last decade to occur by facilitated diffusion... more Anandamide (AEA) uptake has been described over the last decade to occur by facilitated diffusion, but a protein has yet to be isolated. In some cell types, it has recently been suggested that AEA, an uncharged hydrophobic molecule, passively diffuses through the plasma membrane in a process that is not proteinmediated. Since that observation, recent kinetics studies (using varying assay conditions) have both supported and denied the presence of an AEA transporter. In this review, we analyze the current literature exploring the mechanism of AEA uptake and endeavor to explain the reasons for the divergent views. One of the main variables among laboratories is the incubation time of the cells with AEA. Initial kinetics (at time points b 1 min depending upon the cell type) isolate events that occur at the plasma membrane and are most useful to study saturability of uptake and effects of purported transport inhibitors upon uptake. Results with longer incubation times reflect events not only at the plasma membrane but also interactions at intracellular sites that may include enzyme(s), other proteins, or specialized lipid-binding domains. Furthermore, at long incubation times, antagonists to AEA receptors reduce AEA uptake. Another complicating factor in AEA transport studies is the nonspecific binding to plastic culture dishes. The magnitude of this effect may exceed AEA uptake into cells. Likewise, AEA may be released from plastic culture dishes (without cells) in such a manner as to mimic efflux from cells. AEA transport protocols using BSA, similar to the method used for fatty acid uptake studies, are gaining acceptance. This may improve AEA solution stability and minimize binding to plastic, although some groups report that BSA interferes with uptake. In response to criticisms that many transport inhibitors also inhibit the fatty acid amide hydrolase (FAAH), new compounds have recently been synthesized. Following their characterization in FAAH +/+ and FAAH À/À cells and transgenic mice, several inhibitors have been shown to have physiological

Journal of Biological Chemistry, 2012

Background: Transport inhibitors modulate endocannabinoid signaling by inhibiting their uptake th... more Background: Transport inhibitors modulate endocannabinoid signaling by inhibiting their uptake through unknown mechanisms. Results: Effects of transport inhibitors upon endocannabinoid uptake and intracellular trafficking were lost in the absence of fatty acid-binding proteins. Conclusion: Fatty acid-binding proteins are physiological targets of transport inhibitors. Significance: These findings identify drug targets for modulating endocannabinoid signaling. N-Acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acidbinding 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 ␣ (PPAR␣). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPAR␣ 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 PPAR␣ 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. N-Acylethanolamines (NAE) 2 are members of a functionally diverse family of signaling lipids. While the endocannabinoid

Journal of Biological Chemistry, Sep 1, 1994

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a... more Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmucol. 46,791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, a-keto esters, and a-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and a-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 p~ inhibitor and 27.7 p~ anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [sHlCP-5S940 binding to CBRl with a Ki of 0.65 p~. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 p~. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo. A'-Tetrahydrocannabinol, the psychoactive marijuana plantderived cannabinoid, binds to a specific brain cannabinoid receptor, CBRl' (1,2). Arachidonoyl ethanolamide (anandamide) and K05-DA00182 (to A. C. H.) and R03-DA07318 (to D. G. D.) from

Proceedings of the National Academy of Sciences of the United States of America, Apr 14, 2009

Micron, 1979

Low density lipoproteins (LDL) of d = 1.030 to 1.040g/ml from human serum were studied by electro... more Low density lipoproteins (LDL) of d = 1.030 to 1.040g/ml from human serum were studied by electron microscopy. Negatively stained preparations were observed in a scanning transmission electron microscope (STEM) and a conventional transmission microscope equipped with a field emission tip (FE-CTEM). The diameter of LDL was found to be 216/1x 4-22/~. The LDL particle exhibits a substructure consisting of spherical units with a diameter of approximately 20-35/~. As many as 33 of these structures are seen and they appear to be distributed evenly over the LDL particle.

Wiley eBooks, 1989

Quality assurance emit assays for drugs of abuse bonded phase extraction in analytical toxicology... more Quality assurance emit assays for drugs of abuse bonded phase extraction in analytical toxicology gas chromatography/mass spectrometry using tabletop instruments screening with high performance liquid chromatography microcolumn and direct-sample-analysis liquid chromatography high pressure liquid chromatography/mass spectrometry dry reagent chemistries drug of abuse profile - cocaine gas chromatography/mass spectrometry screening for anabolic steroids drug analysis technology - pitfalls and problems of drug testing.

Aaps Journal, Feb 26, 2010

Visual Neuroscience, Nov 1, 2005

There is much evidence for an endocannabinoid system in the retina. However, neither the distribu... more There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC50s of 21 nM and 1.5 μM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20°C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4°C, or preincubated with 100 nM MAFP or 10 μM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

Frontiers in Pharmacology, Oct 13, 2016

biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivati... more biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown.

Journal of Biological Chemistry, Apr 1, 2006

The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2... more The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2H3) has been proposed to occur via a saturable transporter that is blocked by specific inhibitors. Measuring uptake at 25 s, when fatty acid amide hydrolase (FAAH) does not appreciably affect uptake, AEA accumulated via a nonsaturable mechanism at 37°C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37°C. Such apparent saturation can be explained by rate-limited delivery of AEA through an unstirred water layer surrounding the cells (1). In support of this, we observed kinetics consistent with rate-limited diffusion at 0°C. Novel transport inhibitors have been synthesized that are either weak FAAH inhibitors or do not inhibit FAAH in vitro (e.g. UCM707, OMDM2, and AM1172). In the current study, none of these purported AEA transporter inhibitors affected uptake at 25 s. Longer incubation times illuminate downstream events that drive AEA uptake. Unlike the situation at 25 s, the efficacy of these inhibitors was unmasked at 5 min with appreciable inhibition of AEA accumulation correlating with partial inhibition of AEA hydrolysis. The uptake and hydrolysis profiles observed with UCM707, VDM11, OMDM2, and AM1172 mirrored two selective and potent FAAH inhibitors CAY10400 and URB597 (at low concentrations), indicating that weak inhibition of FAAH can have a pronounced effect upon AEA uptake. At 5 min, the putative transport inhibitors did not reduce AEA uptake in FAAH chemical knockout cells. This strongly suggests that the target of UCM707, VDM11, OMDM2, and AM1172 is not a transporter at the plasma membrane but rather FAAH, or an uncharacterized intracellular component that delivers AEA to FAAH. This system is therefore unique among neuro/immune modulators because AEA, an uncharged hydrophobic molecule, diffuses into cells and partial inhibition of FAAH has a pronounced effect upon its uptake.

Clinical Chemistry, Feb 1, 1986

We describe a quantitative screen for hypnotic-sedative drugs in which we use capillarygas chroma... more We describe a quantitative screen for hypnotic-sedative drugs in which we use capillarygas chromatographywith a nitrogen-phosphorus detector (GC/NPD) as the primary methodand capillarygas chromatography-massspectrometry (GC-MS) for confirmation.GC retentiontimes of the acidextracted underivatizeddrugs were stable (CV5 <1%), and the detector response varied linearly over a 20-fold concentrationrange with a mean correlationcoefficientfor 11 drugs of 0.989. The limitsof detectionwere satisfactory(0.5 mg/Lin a 0.5-mL serum sample and 1-L injectionvolume), as were precision (average CV 5.2% within day, 6.4% betweenday).

Vitamins, Jun 25, 2013

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (a... more Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of Nacylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and-independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg 2+-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-Nacylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.

Cns & Neurological Disorders-drug Targets, Mar 1, 2013

Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally refer... more Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally referred to as Nacylethanolamines (NAEs). NAEs include anti-inflammatory and analgesic palmitoylethanolamide, anorexic oleoylethanolamide, and the endocannabinoid anandamide. Since the endogenous levels of NAEs are principally regulated by enzymes responsible for their biosynthesis and degradation, these enzymes are expected as targets for the development of therapeutic agents. Thus, a better understanding of these enzymes is indispensable. The classic "N-acylationphosphodiesterase pathway" for NAE biosynthesis is composed of two steps; the formation of Nacylphosphatidylethanolamine (NAPE) by N-acyltransferase and the release of NAE from NAPE by NAPE-hydrolyzing phospholipase D (NAPE-PLD). However, recent studies, including the analysis of NAPE-PLD-deficient (NAPE-PLD-/-) mice, revealed the presence of NAPE-PLD-independent multi-step pathways to form NAEs from NAPE in animal tissues. Our recent studies using NAPE-PLD-/mice also suggest that NAE is formed not only from NAPE, but also from Nacylated plasmalogen-type ethanolamine phospholipid (N-acyl-plasmenylethanolamine) through both NAPE-PLDdependent and-independent pathways. Here, we present recent findings on NAE biosynthetic pathways mainly occurring in the brain.

Perspectives in Biology and Medicine, 1977