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PubMed1998

Effects of the farnesyltransferase inhibitor UCF-1C/manumycin on growth and p21-ras post-translational processing in NIH3T3 cells.

Examination of the effect of the farnesylprotein transferase (FPTase) inhibitor UCF1-C/manumycin on NIH3T3 cells transfected with a normal N-ras gene and expressing high levels of the corresponding p21-ras protein showed that 10 microm UCF1-C immediately and reversibly inhibited growth in these cells, without modifying cell-death rate, thus acting as a cytostatic. There was also a 98% reduction of p21-ras neofarnesylation and a 3-fold decrease in total content in p21-ras products, yet without gross modification of the relative content in the post-translational products and without accumulation of the native protein to detectable levels. UCF1-C likewise reversibly inhibited growth in parental NIH3T3 cells, as well as in sub-strains expressing a transfected normal or mutated H-ras gene. Together with the fact that the well-developed network of actin stress fibers present in the NIH3T3 (N-ras) cells was not affected by the FPTase inhibitor, these data indicate that its growth-inhibitory effect is not necessarily in direct relation with that exerted on p21-ras processing. Alternatively, it might be causally related to the decreased prenylation of other cellular proteins, perhaps included among the 13 proteins, unrelated to p21-ras, of which the farnesylation was also reduced under UCF1-C treatment. Some cells transformed by a ras or non-ras oncogene might exhibit higher susceptibility towards FPTase inhibitors than normal cells, but this might then be attributable to differences in the pattern of expression and/or in the functional importance of non-ras farnesylated proteins.

3T3 Cells↗
PubMed1998

Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: comparison of immunocytochemical data with biochemical measurements.

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF-7 cells under estrogenic and anti-estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER-EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E2 and DES) and pure anti-estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4-OH-TAM and RU 39,119). E2-induced ER down-regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down-regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre-labeling of the cells with [3H]TAZ indicated that all investigated ligands eliminate pre-existing ER through binding to newly synthetized receptors, since [3H]TAZ co-valently associates with ER; E2 and RU 58,668 were more effective than 4-OH-TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre-exposed to [3H]TAZ. Nuclear location of [3H]TAZ-ER complexes may explain this discrepancy, since pure AE-ER complexes were reported to be incapable of nuclear translocation.

Breast Neoplasms↗
PubMed1999

Apoptosis of medulloblastoma cells in vitro follows inhibition of farnesylation using manumycin A.

Medulloblastoma is a malignant cerebellar tumor usually manifesting in childhood. We have previously shown that blocking the mevalonate pathway with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits medulloblastoma proliferation and induces apoptosis in vitro. The underlying mechanism may involve blocking post-translational modification of important mitogenic signal-transduction proteins. We show that p21 ras processing is blocked by lovastatin, suggesting that inhibition of isoprenylation may be important in lovastatin-induced apoptosis. To test this hypothesis, manumycin A, an antibiotic which inhibits farnesyl protein transferase and thus farnesylation, was administered to 4 medulloblastoma cell lines in vitro. We found that blocking protein farnesylation with manumycin A was followed by apoptosis in a time- and dose-dependent manner. However, cell death induced by manumycin A was uniformly more rapid and efficient, requiring only 12 to 24 hr of treatment, than lovastatin-induced apoptosis, which required 36 to 96 hr (depending on the cell line tested). In addition, unlike lovastatin, which caused cell-cycle arrest in G1 phase and HMG-CoA reductase gene up-regulation, manumycin A had no effect on the cell cycle and resulted in down-regulation of HMG-CoA reductase gene expression. In both lovastatin- and manumycin A-treated cells, cellular cysteine protease precursor (CPP32) was activated, confirming the occurrence of apoptosis.

Antineoplastic Agents↗
PubMed1997

Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme activity that degrades neuromodulatory fatty acid amides, including oleamide and anandamide. A single 2.5-kb FAAH mRNA is distributed throughout the rat CNS and accumulates progressively between embryonic day 14 and postnatal day 10, remains high until postnatal day 30, then decreases into adulthood. FAAH enzymatic activity, as measured in dissected brain regions, was well correlated with the distribution of its messenger RNA. In situ hybridization revealed profound distribution of FAAH mRNA in neuronal cells throughout the CNS. The most prominent signals were detected in the neocortex, hippocampal formation, amygdala, and cerebellum. The FAAH distribution in the CNS suggests that degradation of neuromodulatory fatty acid amides at their sites of action influences their effects on sleep, euphoria, and analgesia.

Amidohydrolases↗
PubMed2000

Role of insulin-like growth factor-I in regulating estrogen receptor-alpha gene expression.

The role of insulin-like growth factor-I (IGF-I) in regulating estrogen receptor-alpha (ER-alpha) gene expression and activity was investigated in the human breast cancer cell line MCF-7. Treatment of cells with 40 ng/ml IGF-I resulted in a 60% decrease in ER-alpha protein concentration by 3 h, and the amount of ER-alpha remained suppressed for 24 h. A multiple-dose ligand-binding assay demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease of 50% in estradiol-binding sites with no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the absence of IGF-I (K(d) = 3 x 10(-10) +/- 0.5 x 10(-10) M) was similar to the dissociation constant in the presence of IGF-I (K(d) = 6 x 10(-10) +/- 0.3 x 10(-10) M). The decrease in ER-alpha protein concentration was paralleled by an 80% decrease in the steady-state amount of ER-alpha mRNA by 3 h. The IGF-I induced decrease in ER-alpha mRNA was due to the inhibition of ER-alpha gene transcription. When an 128-base pair ER-alpha-promoter-CAT construct was transfected into MCF-7 cells, treatment with IGF-I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER-alpha, treatment with IGF-I induced two endogenous estrogen-regulated genes, progesterone receptor and pS2, by 4- and twofold, respectively. The pure antiestrogen ICI-164, 384 blocked this induction, suggesting that ER-alpha mediates the effects of IGF-I. Transient co-transfections of wild-type ER-alpha and an estrogen response element-CAT reporter into COS-1 cells demonstrated that IGF-I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3-kinase inhibitors blocked the IGF-I effects on ER-alpha expression and activity, suggesting that these kinases may be involved in the cross-talk between the IGF-I and ER-alpha pathways.

Animals↗
PubMed2000

Localization of cannabinoid CB(1) receptor mRNA in neuronal subpopulations of rat striatum: a double-label in situ hybridization study.

Double-label in situ hybridization was used to identify the phenotypes of striatal neurons that express mRNA for cannabinoid CB(1) receptors. Simultaneous detection of multiple mRNAs was performed by combining a (35)S-labeled ribonucleotide probe for CB(1) mRNA with digoxigenin-labeled riboprobes for striatal projection neurons (preprotachykinin A, prodynorphin, and preproenkephalin mRNAs) and interneurons (vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), somatostatin, and glutamic acid decarboxylase (Mr 67,000; GAD67) mRNAs). To ascertain whether CB(1) mRNA was a marker for striatal efferents, digoxigenin-labeled probes for mRNA markers of both striatonigral (prodynorphin or preprotachykinin A mRNAs), and striatopallidal (proenkephalin mRNAs) projection neurons were combined with the (35)S-labeled probe for CB(1). A mediolateral gradient in CB(1) mRNA expression was observed at rostral and mid-striatal levels; in the same coronal sections the number of silver grains per cell ranged from below the threshold of detectability at the medial and ventral poles to saturation at the dorsolateral boundary bordered by the corpus callosum. At the caudal level examined, CB(1) mRNA was denser in the ventral sector relative to the dorsal sector. Virtually all neurons expressing mRNA markers for striatal projection neurons colocalized CB(1) mRNA. Combining a (35)S-labeled riboprobe for CB(1) with digoxigenin-labeled riboprobes for both preproenkephalin and prodynorphin confirmed localization of CB(1) mRNA to striatonigral and striatopallidal neurons expressing prodynorphin and preproenkephalin mRNAs, respectively. However, CB(1) mRNA-positive cells that failed to coexpress the other markers were also apparent. CB(1) mRNA was localized to putative GABAergic interneurons that express high levels of GAD67 mRNA. These interneurons enable functional interactions between the direct and indirect striatal output pathways. By contrast, aspiny interneurons that express preprosomatostatin mRNA and cholinergic interneurons that coexpress ChAT and VAChT mRNAs were CB(1) mRNA-negative. The present data provide direct evidence that cannabinoid receptors are synthesized in striatonigral neurons that contain dynorphin and substance P and striatopallidal neurons that contain enkephalin. By contrast, local circuit neurons in striatum that contain somatostatin or acetylcholine do not synthesize cannabinoid receptors. Published 2000 Wiley-Liss, Inc.

Animals↗
PubMed1996

The uterus is a potential site for anandamide synthesis and hydrolysis: differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period.

Arachidonoylethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors. We demonstrated previously that ligand-receptor signaling with cannabinoids is operative in both the mouse embryo and uterus during the periimplantation period. In the present investigation, we provide evidence that mouse uterus has the enzymatic capacities to form (synthase) and hydrolyze (amidase) anandamide. These activities were primarily localized in uterine microsomes and were dependent upon pH, time, protein, and substrate concentrations. The rate of formation of anandamide was dependent on arachidonic acid (Km: 3.8 microM and Vmax: 2.5 nmol/h/mg protein) and ethanolamine (Km:1.2 mM and Vmax:4.1 nmol/h/mg protein) concentrations. The amidase activity showed an apparent Km of 67 microM and Vmax of 3.5 nmol/min/mg protein with anandamide as a substrate. While the synthase showed maximal activity at pH 9.0, the amidase activity was maximal at pH 8.5. As reported previously, phenylmethylsulfonyl fluoride (PMSF) or arachidonyl trifluoromethyl ketone (ATK) inhibited the amidase activity in a dose-dependent manner. In contrast, PMSF was not inhibitory to synthase activity, rather it stimulated synthase activity at lower concentrations. Further, inhibitory effects of ATK were only modest toward the synthase activity and the effects were not concentration-dependent. To determine whether uterine synthase and/or amidase activity have any physiological significance with respect to uterine receptivity and implantation during early pregnancy, profiles of synthase and amidase activities were analyzed in mouse uterine microsomes obtained during early pregnancy or pseudopregnancy. It should be noted that the synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state are critical to the embryo implantation process. In the mouse, the uterus becomes receptive for implantation only for a limited period during pregnancy or pseudopregnancy. The uterus becomes receptive on day 4 (the day of implantation) and by day 5, it becomes nonreceptive for blastocyst implantation (Paria et al., 1993: Proc Natl Acad Sci USA 90:10159-10162.). Both anandamide synthase and amidase activities remained virtually unaltered on days 1-4 of pregnancy. In contrast, while the synthase activity increased, the amidase activity decreased in the uterus on day 5 of pseudopregnancy (nonreceptive phase) as compared to those observed on day 4 of pregnancy or pseudopregnancy (receptive phase). The synthase and amidase activities in surgically separated implantation and interimplantation sites showed an interesting profile on days 5-7 of pregnancy; the synthase activity was lower in implantation sites as compared to that in interimplantation sites. In contrast, amidase activity was higher in implantation sites compared with that in interimplantation sites. Since we have shown previously that cannabinoids including anandamide interfere with preimplantation mouse embryo development, the local modulation of anandamide formation and hydrolysis by the implanting blastocysts could be critical for successful embryonic growth, implantation, and pregnancy establishment. The finding of increased synthase activity with concomitant decrease in amidase activity in the uterus on day 5 of pseudopregnancy, when the uterus in hostile to blastocyst survival and implantation, is consistent with this assumption. Further indomethacin, known to interfere with arachidonate metabolism and embryo implantation, stimulated the synthase activity, while inhibiting the amidase activity in the uterus in vivo and in vitro. Finally, considering the kinetics and profiles of these two enzymatic reactions during early pregnancy, the results suggest that synthase and amidase may be two separate enzymes in the mouse uterus. This investigation constitutes the first detailed studies on anandamide synthase and amidase activities in the female reproductive t

Amidohydrolases↗
PubMed2000

Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection.

A fluorimetric determination method for N-arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled-column high-performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4-N-chloroformylmethyl-N-methylamino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Emporetrade mark), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 x 4.6 mm i.d. ) and octadecylsilica columns (250 x 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3. 37 +/- 0.73 pmol/g with 6.47 and 3.57% of intra- and inter-day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16-fold higher than that of rats without PMSF (p < 0.01).

Animals↗
PubMed2000

Expression of connexin 30 in Xenopus embryos and its involvement in hatching gland function.

Connexins are a family of proteins that assemble to form gap junction channels. Cell-cell communication through gap junctions mediates many important events in embryogenesis, including limb patterning, lens physiology, neuronal function, left-right asymmetry, and secretion from gland tissue. We studied the expression of connexin 30 (Cx30) in the Xenopus embryo and find that it is expressed in the developing hatching gland and pronephros. To determine whether its expression plays a functional role in the activity of the hatching gland, we exposed pre-hatching embryos to drugs that block gap junctional communication. This resulted in a continuation of normal growth and development but specifically abolished hatching. The treatment did not affect Cx30 or Xenopus hatching enzyme transcription, suggesting a post-transcriptional effect on Cx30 gap junctions. We conclude that junctional communication, possibly mediated by Cx30, is involved in secretion of hatching enzyme in Xenopus.

Administration, Topical↗
PubMed2000

Early migratory rat neural crest cells express functional gap junctions: evidence that neural crest cell survival requires gap junction function.

Gap junctions mediate crucial intercellular interactions during development. This study provides evidence that early migrating rat neural crest cells assemble functional gap junctions, as demonstrated by dye transfer following microinjection of single cells, which were phenotypically identified as neural crest cells by their expression of the low- affinity nerve growth factor receptor. An immunohistochemical analysis using connexin- specific antibodies revealed that migrating rat neural crest cells express the gap junction constituents connexins 43 (Cx 43) and Cx 46. We tested the hypothesis that gap junctions play an important role during early neural crest cell development by perturbing their function in migrating neural crest cells. Our data show that markedly decreasing gap junction communication between these neural crest cells in vitro with either 18alpha-glycyrrhetinic acid or anandamide decreases their survival, whereas oleamide, a less effective blocker of connexon function, had quantitatively less effect on neural crest cell death. This cell death was associated with the occurrence of DNA nicking as detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) procedure, suggesting cell death via apoptosis. The effect of 18alpha-glycyrrhetinic acid and anandamide on neural crest cell survival was reversible and was not mimicked by the structurally related compounds glycyrrhizic acid and palmitoylethanolamide, respectively, which do not uncouple cells. These results indicate that gap junctions are necessary for the survival of spinal neural crest cells.

1-Octanol↗
PubMed2000

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats.

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.

Alkaloids↗
PubMed2003

Functional morphology and physiology of slowly adapting pulmonary stretch receptors.

Since the original work by Hering and Breuer (1868) on slowly adapting pulmonary stretch receptors (SARs), numerous studies have demonstrated that these receptors are the lung vagal afferents responsible for eliciting the reflexes evoked by moderate lung inflation. SARs play a role in controlling breathing pattern, airway smooth muscle tone, systemic vascular resistance, and heart rate. Both anatomical and physiological studies support the contention that SARs, by their close association with airway smooth muscle, continuously sense the tension within the myoelastic components of the airways caused by lung inflation, smooth muscle contraction, and/or tethering of small intrapulmonary airways to the lung parenchyma. As a result, the receptor field location within the tracheobronchial tree of a SAR plays an important role in its discharge pattern, with variations in airway transluminal pressure and airway smooth muscle orientation being important modulating factors. The disruption of airway myoelastic components in various pulmonary diseases would be expected to alter the discharge pattern of SARs, and contribute to changes in breathing pattern and airway smooth muscle tone.

Adaptation, Physiological↗
PubMed2003

Functional morphology and physiological properties of bronchopulmonary C-fiber afferents.

Nonmyelinated (C-) fibers represent the majority of vagal afferents innervating the airways and lung, and play an important role in regulating the respiratory and cardiovascular functions under both normal and abnormal physiologic conditions. Studies of the relationship between the conduction velocities of the vagal afferents and their sensitivities to capsaicin and other chemical irritants reveal that C-fibers are the primary type of chemosensitive afferents in the rat lung. Furthermore, a distinct sensitivity to capsaicin and a weak response to lung inflation are the defining characteristics of these afferents. In cultured rat nodose and jugular ganglion neurons, capsaicin-sensitive cells were identified by measurement of the capsaicin-evoked calcium transients using the Fura-2-based ratiometric imaging technique. The percentage of capsaicin-sensitive neurons gradually decreases as the cell diameter increases. However, the capsaicin-sensitive neurons cannot be precisely identified solely on the basis of the cell size. Anandamide, an endogenous cannabinoid released from leukocytes and epithelial cells, consistently evokes a stimulatory effect on pulmonary C-fiber endings by activating vanilloid receptor type 1 (VR1). The discharge pattern of pulmonary C-fibers evoked by anandamide closely resembles that produced by a much lower ( approximately 1/600) dose of capsaicin in the same fibers. Whether anandamide acts as a potential endogenous ligand to VR1 at the C-fiber terminals is unclear, and the physiological role of VR1 in modulating the transduction properties of these afferents also remains to be determined.

Afferent Pathways↗
PubMed2003

Botanical insecticides for controlling agricultural pests: piperamides and the Colorado Potato Beetle Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae).

The efficacy of extracts from two Piperaceae species, Piper nigrum L. and P. tuberculatum Jacq. were evaluated using larvae and adults of the Colorado Potato Beetle Leptinotarsa decemlineata (Say). Young larvae and neonates were the most susceptible; a 24-h LD(50) of 0.064% extract of P. tuberculatum was determined for 4-day-old larvae, while 0.05% extract of P. nigrum reduced larval survival up to 70% within one week after treatment of potato Solanum tuberosum L. (Solanaceae) plants. When an insecticide resistant strain of L. decemlineata larvae was tested with the P. tuberculatum extract, there was less than a 2-fold tolerance ratio compared to the 22-fold tolerance ratio to cypermethrin, a pyrethroid. Older larvae, pre-pupal stage and adults, were less sensitive to the P. nigrum extracts; the 24-h LD(50) was 0.5% (95% C.I. = 0.36, 0.65). However, the same concentration was equally effective under field conditions. In the greenhouse, P. nigrum at 0.5% was as effective at reducing adult L. decemlineata feeding as combinations with 2 separate botanical mixtures, garlic and lemon grass oil. Under field conditions, the residual activity of the P. nigrum extracts was less than 3 h. When adult L. decemlineata were placed on treated plants exposed to full sunlight for 0, 1.5, and 3 h, leaf damage progressively increased as the main active compound, piperine, was found to degrade by 80% after 3 h. An in vitro polysubstrate monoxygenase (PSMO) enzyme assay, using the substrate methoxyresorufin O-demethylation (MROD), determined that the principal P. nigrum active compound, piperine, is responsible for inhibition of that specific enzyme. The results suggest that Piper extracts could be used effectively as contact botanical insect control agents to protect potato plants from developing L. decemlineata larvae at concentrations less than 0.1%. There is also potential for Piper extracts to control insecticide resistant populations in conjunction with other integrated pest management (IPM) strategies used in conventional and organic agriculture.

Agriculture↗
PubMedDate not supplied

High-performance liquid chromatography and fluorometric detection of arachidonylethanolamide (anandamide) and its analogues, derivatized with 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulp honyl-2,1 ,3- benzoxadiazole (DBD-COCl).

The endogeneous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide) and its analogues, oleinylethanolamide, palmitylethanolamide and eicosapentaenoylethanolamide, were derivatized with a fluorogenic reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminsulpho ny1-2,1,3- benzoxadiazole (DBD-COCl). They were separated on a reversed phase HPLC with a mobile phase of acetonitrile:water. The fluorometric detection of the derivatives was made at 560 nm with excitation at 450 nm and the detection limits for anandamide was 20 fmol on column. The structures of DBD-CO-ethanolamides were confirmed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS).

Arachidonic Acids↗
PubMed2006

Determination of the endocannabinoid anandamide in human plasma by high-performance liquid chromatography.

Anandamide (N-arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole (DBD-COCl), the analyte was separated using an acetonitrile-water gradient at a flow rate of 0.8 mL/min, and spectrophotometric detection at 560 nm with an excitation wavelength of 450 nm. The retention times for anandamide and R+-methanandamide (internal standard) were 27.1 and 30.7 min, respectively. The validated quantification range was 1-15 ng/mL. The developed procedure was applied to determine anandamide levels in human plasma following a 24 h incubation of human whole blood at 37 degrees C in the presence or absence of phenylmethylsulfonyl fluoride, an inhibitor of the anandamide-degrading enzyme fatty acid amide hydrolase. Anandamide levels determined under both conditions were within the validated concentration range with anandamide levels being 2.3-fold higher in plasma from PMSF-treated blood.

Arachidonic Acids↗