Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125 Ileptin-serum complex demonstrated 125 I-leptin eluting in significant proportion associated with macromolecules. The 125 I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED 50 : 0.73 Ϯ 0.09 nM, mean Ϯ SEM, n ϭ 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80-or 100-kD proteins could be the soluble leptin receptor as ‫ف‬ 10% of the bound 125 I-leptin was immunoprecipitable with leptin receptor antibodies.Significantly higher ( P Ͻ 0.001) proportions of total leptin circulate in the bound form in lean (46.5 Ϯ 6.6%) compared with obese (21.4 Ϯ 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean ( P Ͻ 0.0005) and three obese ( P Ͻ 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 Ϯ 1.9 vs. 1.3 Ϯ 0.4 ng/ml) compared with those in obese subjects (28.3 Ϯ 9.8 vs. 14.7 Ϯ 5.3). No significant ( P Ͼ 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2-to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [14 C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.Following the discovery of two G protein-coupled receptors, CB1 and CB2, which respond to ⌬ 9 -tetrahydrocannabinol, the active principal in marijuana, a search was initiated to identify endogenous ligands for these receptors. Anandamide (N-arachidonyl ethanolamide) was the first endocannabinoid discovered in 1992 by screening porcine brain extracts for compounds that bound to the cannabinoid receptor (Devane et al., 1992). It was later shown that anandamide could stimulate cannabinoid receptor-mediated signal transduction (Felder et al., 1993). The second endocannabinoid identified was 2-arachidonoyl glycerol (2-AG), which was isolated from canine gut and shown to have in vivo effects similar to ⌬ 9 -tetrahydrocannabinol (Mechoulam et al., 1995). Very recently, a third endocannabinoid, noladin ether, was also isolated from porcine brain (Hanus et al., 2001). Both anandamide and 2-AG are agonists at both the CB1 and CB2 receptors. Noladin ether has been shown to bind to the CB1 receptor with nanomolar affinity and to the CB2 receptor with low micromolar affinity, but functional activity has not yet been determined (Hanus et al., 2001).In the course of development of a bioanalytical method to measure anandamide in brain and peripheral tissues and brain microdialysate, a second analyte was seen that had the same molecular weight as anandamide but a shorter retention time, and therefore, could not be anandamide (Fig. 1). The peak was hypothesized to be O-arachidonoyl ethanolamine, and an authentic standard was subsequently synthesized (BIOMOL Research Laboratories, Plymouth Meeting, PA). Based on its chromatographic and mass spectrometric properties compared with the synthesized standard, the unknown analyte was confirme...

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.Key words: Anandamide; Cannabis; Cannabinoid receptor; Marijuana porcine brain and found to be a lipid of novel structure [7]. Anandamide displayed specific binding to the CBI receptor and inhibited a prototypical twitch response in mouse vas deferens. Anandamide has also been shown to induce similar behavioral [8,9], pharmacological [10,11], and signal transduction effects [12] as classical cannabinoid agonists, but high concentrations were required to induce these effects. Levels of anandamide were first estimated to occur at 0.4 pmol/g (133 pg/g) in whole porcine brain [7], and recently quantitated in porcine and bovine brain at 173 pmol/g (60 ng/g) and 101 pmol/g (35 ng/g) respectively [13]. A recent study reports levels of anandamide in rat testis to be considerably lower (6 pmol/ g, 2.1 ng/g) [14]. However, anandamide has never been isolated from human tissue or fluids. Furthermore, levels of anandamide have not been measured in regions of rat brain or in tissues such as spleen where CB2 receptors have been shown to be expressed at high levels. Studies of anandamide distribution should help elucidate the physiologic role of anandamide as a cannabimimetic eicosanoid and possibly broader functions. In this study we report the isolation and quantitation of anandamide by liquid chromatography/mass spectrometry in various tissues and fluids from postmortem human and rat.

The deposition of the ␤ amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid ␤-protein of 39 -43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP.We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease.When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid ␤-protein peptide and shows a reduction of residues 17-42 of A␤ (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.The generation of the ␤ amyloid peptide is thought to be the result of processing of the amyloid precursor protein (APP) 1 by one or more proteases. After the deduced amino acid sequence of APP was revealed, a number of laboratories initiated studies to purify and characterize the N-terminal cleaving enzyme of amyloid ␤-protein (A␤), termed ␤-secretase (1). The cleavage of the Met 596 -Asp 597 bond of the full-length APP generates the N-terminal amino acid of A␤, which was first shown by Glenner and Wong (2) to be aspartic acid. ␤-Secretase is yet an unidentified protease.Several themes and strategies influenced the direction of investigation of ␤-secretase. The first strategy was to follow a traditional biochemical purification. Assays were utilized in which short peptide substrates were substituted for the large transmembrane precursor protein (1). Any enzyme capable of making a methionine (M)/aspartic acid (D) cleavage could be designated a potential ␤-secretase. The second theme, since the amino acid that surrounded the N terminus of A␤ was found to be a methionine, was some laboratories have rationalized that a search for an enzyme with chymotrypsin-like specificity (a specificity for cleavage of subtrates containing a neutral hydrophobic residue at the S1 subsite) was necessary (3-7).To facilitate the second approach, we have developed a method to identify chymotrypsin-l...

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