Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells.
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Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression. (+info)
On the interaction of a lipophilic drug with different sites of rat-liver microsomes. Equilibrium studies with a substituted pleuromutilin.
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The binding of the diterpenoid drug 14-deoxy-14-[(2'-diethylamino-ethyl)-mercaptoacetoxy]-dihydromutilin hydrogen fumarate in the cell of rat liver is mainly to the microsomal fraction. Besides specific binding to cytochrome P-450, where the enzymic degradation of the drug occurs, we observed a very high number of identical sites (site A) with an affinity of approximately 4.2 x 10(3) M(-1) (25 degrees C, PH 7.4). Model investigations demonstrate that these interactions occur almost exclusively with the microsomal phospholipid moiety. Their capacity for the drug was determinated to be of the order of 0.2 mol/mol phospholipid. The specific interaction of the pleuromutilin derivative with cytochrome P-450 gives rise to different spectral changes of the protein. At low concentrations where weak cooperativity of the overall binding to microsomes (sites B) was found, the formation of a type I complex was observed. At increasing concentrations of the drug this interaction vanishes and a spectral change of a different type (modified type II) arises. The affinity for this complex is identical with that of the phospholipid binding sites. The interaction of the drug with the phospholipid moiety might give rise to dual effects. Firstly the very near neighbourhood of a multitude of relatively weak binding sites will facilitate a transport of the drug along the microsomal membranes. Secondly, the loading of the membranes with the drug at high concentrations might influence the binding to cytochrome P-450 so that a qualitatively different interaction takes place. (+info)
Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.
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1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12 - 18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of approximately 5. Homologues with chain lengths < or = eight carbon atoms gave < 20% inhibition at 100 microM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 microM) and palmitoylisopropylamide (30 and 100 microM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB(1) or CB(2) receptors. (+info)
Vitreous structure, IV. Chemical composition of the insoluble residual protein fraction from the rabbit vitreous.
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Analysis of the structural proteins in the rabbit vitreous showed that the hydroxyproline content was 3.1 per cent w/w compared to a value of 10.9 per cent w/w for equivalent samples obtained from cattle. In contrast to the discreet fibers in bovine vitreous, the rabbit constituents occur as an aggregate of fibrils with a diameter of 15 to 20 a. The amino acid and carbohydrate composition was similar to vascular basement membrane and isolated fractions contained significant amounts of palmitic and stearic acids. The data indicate that the variability of vitreous structure in different species is not only quantitative, but also qualitative. It is suggested that in the rabbit the structural proteins may be derived primarily from the atrophied hyaloid system and that little, if any, secondary vitreous formation occurs in this animal. (+info)
Structural specificity in ether lipid biosynthesis. Formation of hydroxyalkyl and oxoalkyl glycerophosphatides.
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Structural specificity of alkyl glycerolipid formation was studied in myelinating rat brain. 1,2-[2-14-C,2-3-H]Heptadecanediol, 1,2-[1-14-C]octadecanediol, and 1-O-2-hydroxy-[1-14-C]heptadecyl-rac-glycerol were administered intracerebrally and their incorporation into phospholipids was determined after time periods ranging from 6 to 48 hours. Experimental evidence is presented to support the following conclusions. (a) Long chain 1,2-alkanediols serve as direct precursors of the 2-hydroxyalkyl moieties of both choline and ethanolamine glycerophosphatides. (b) 1,2-Alkanediols are oxidized to 1-hydroxy-2alkanones which serve as precursors of the 2-oxoalkyl moieties of the glycerophosphatides. (c) Oxidations of hydroxy groups and reductions of oxo groups do not occur at the phospholipids, but are confined to 1,2-alkanediols and 1-hydroxy-2-alkanones, respectively. (d) Metabolic degradation of 1,2-alkanediols proceeds by oxidation to the corresponding 2-hydroxy and/or 2-oxo fatty acids and decarboxylation. (+info)
Polarized targeting of peripheral membrane proteins in neurons.
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Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins. (+info)
The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.
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1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo. (+info)
The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.
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The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate. (+info)