Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1.
(25/954)
The vanilloid receptor type 1 (VR1) is a heat-activated ionophore preferentially expressed in nociceptive neurons of trigeminal and dorsal root ganglia (DRG). VR1, which binds and is activated by capsaicin and other vanilloid compounds, was noted to interact with the endocannabinoid anandamide (ANA) and certain inflammatory metabolites of arachidonic acid in a pH-dependent manner. At pH < or = 6.5 ANA induced (45)Ca(2+) uptake either in primary cultures of DRG neurons or cells ectopically expressing C-terminally tagged recombinant forms of VR1 with an EC(50) = approximately 10 microm at pH 5.5. Capsazepine, a potent antagonist of vanilloids, inhibited ANA-induced Ca(2+) transport in both cell systems. Vanilloids displaced [(3)H]ANA in VR1-expressing cells, suggesting competition for binding to VR1. Ratiometric determination of intracellular free calcium and confocal imaging of the VR1-green fluorescent fusion protein revealed that, at low pH (< or =6.5), ANA could induce an elevation of intracellular free Ca(2+) and consequent intracellular membrane changes in DRG neurons or transfected cells expressing VR1. These actions of ANA were similar to the effects determined previously for vanilloids. The ligand-induced changes in Ca(2+) at pH < or = 6.5 are consistent with the idea that ANA and other eicosanoids act as endogenous ligands of VR1 in a conditional fashion in vivo. The pH dependence suggests that tissue acidification in inflammation, ischemia, or traumatic injury can sensitize VR1 to eicosanoids and transduce pain from the periphery. (+info)
Receptor-independent effects of natural cannabinoids in rat peritoneal mast cells in vitro.
(26/954)
Cannabinoids can activate CB(1) and CB(2) receptors. Since a CB(2) mRNA has been described in rat peritoneal mast cells (RPMC), we investigated a series of cannabinoids and derivatives for their capacity to stimulate RPMC. Effects of natural cannabinoids Delta(9)-tetrahydrocannabinol (Delta(9)-THC), Delta(8)-THC, endocannabinoids (anandamide, palmitoylethanolamide) and related compounds (N-decanoyl-, N-lauroyl-, N-myristoyl-, N-stearoyl- and N-oleoyl-ethanolamines; N-palmitoyl derivatives (-butylamine, -cyclohexylamine, -isopropylamine); and N-palmitoyl, O-palmitoylethanolamine), and synthetic cannabinoids including WIN 55,212-2, SR141716A and SR144528 were assessed for their capacity to induce histamine release or prime RPMC stimulated by compound 48/80. Only Delta(9)-THC and Delta(8)-THC could induce non-lytic, energy- and concentration-dependent histamine releases from RPMC (respective EC(50) values: 23.5+/-1.2; 53.4+/-20.6 microM, and maxima: 71.2+/-5.5; 55.7+/-2.7% of the total RPMC histamine content). These were not blocked by CB(1) (SR141716A) or CB(2) (SR144528) antagonists, but reduced by pertussis toxin (100 ng/ml). Endocannabinoids and analogues did neither induce histamine secretion, nor prime secretion induced by compound 48/80 (0.2 microg/ml). Delta(9)-THC and Delta(8)-THC induced in vitro histamine secretion from RPMC through CB receptor-independent interactions, partly involving G(i/o) protein activation. (+info)
Administration of endocannabinoids prevents a referred hyperalgesia associated with inflammation of the urinary bladder.
(27/954)
BACKGROUND: Referred hyperalgesia to a somatopically appropriate superficial site is a cardinal symptom of visceral inflammatory pain and has been demonstrated after turpentine-induced urinary bladder inflammation in the rat. The authors examined the effect of the endocannabinoids anandamide and palmitoylethanolamide on the referred hyperalgesia associated with this model. METHODS: After measurement of baseline limb withdrawal latencies to a noxious heat stimulus, the bladders of 50 female Wistar rats were inflamed by intravesical administration of 0.5 ml 50% turpentine. Ten or 25 mg/kg of anandamide or palmitoylethanolamide or vehicle were administered immediately before introduction of turpentine. Antagonists to both the cannabinoid CB1 and CB2 receptors were coadministered with the higher dose of endocannabinoids. Latencies were recorded 2, 4, 6, 8, and 24 h after removal of turpentine. The difference between forelimb and hind limb withdrawal latencies was plotted against time, and areas under these curves were compared. RESULTS: Inflammation of the urinary bladder was associated with a relative thermal hyperalgesia referred to the hind limb. Anandamide and palmitoylethanolamide attenuated this referred hyperalgesia at doses of 10 and 25 mg/kg. The CB1 receptor antagonist SR141716A reduced the antihyperalgesic effect of anandamide, but the CB2 antagonist SR144528 did not. Coadministration of SR141716A with palmitoylethanolamide did not affect the antihyperalgesic effect but was reduced by SR144528. CONCLUSIONS: Anandamide (via CB1 receptors) and palmitoylethanolamide (putatively via CB2 receptors) attenuated a referred hyperalgesia in a dose-dependent fashion. CB1 and CB2 receptors are strategically situated to influence the nerve growth factor-driven referred hyperalgesia associated with inflammation of the urinary bladder. These data implicate cannabinoids as a novel treatment for vesical pain. (+info)
Progesterone up-regulates anandamide hydrolase in human lymphocytes: role of cytokines and implications for fertility.
(28/954)
Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-12 and IFN-gamma reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy. (+info)
Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol.
(29/954)
The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry. (+info)
Mechanisms of endocannabinoid inactivation: biochemistry and pharmacology.
(30/954)
The endocannabinoids, a family of endogenous lipids that activate cannabinoid receptors, are released from cells in a stimulus-dependent manner by cleavage of membrane lipid precursors. After release, the endocannabinoids are rapidly deactivated by uptake into cells and enzymatic hydrolysis. Endocannabinoid reuptake occurs via a carrier-mediated mechanism, which has not yet been molecularly characterized. Endocannabinoid reuptake has been demonstrated in discrete brain regions and in various tissues and cells throughout the body. Inhibitors of endocannabinoid reuptake include N-(4-hydroxyphenyl)-arachidonylamide (AM404), which blocks transport with IC50 (concentration necessary to produce half-maximal inhibition) values in the low micromolar range. AM404 does not directly activate cannabinoid receptors or display cannabimimetic activity in vivo. Nevertheless, AM404 increases circulating anandamide levels and inhibits motor activity, an effect that is prevented by the CB1 cannabinoid antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole -3-carboxamide hydrochloride (SR141716A). AM404 also reduces behavioral responses to dopamine agonists and normalizes motor activity in a rat model of attention deficit hyperactivity disorder. The endocannabinoids are hydrolyzed by an intracellular membrane-bound enzyme, termed anandamide amidohydrolase (AAH), which has been molecularly cloned. Several fatty acid sulfonyl fluorides inhibit AAH activity irreversibly with IC50 values in the low nanomolar range and protect anandamide from deactivation in vivo. alpha-Keto-oxazolopyridines inhibit AAH activity with high potency (IC50 values in the low picomolar range). A more thorough characterization of the roles of endocannabinoids in health and disease will be necessary to define the significance of endocannabinoid inactivation mechanisms as targets for therapeutic drugs. (+info)
Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo.
(31/954)
Prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) are generated by the action of cyclooxygenase-2 on the endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, respectively. These novel eicosanoids may have unique pharmacological properties and/or serve as latent sources of prostaglandins at sites remote from their tissue of origin. Therefore, we investigated the metabolism of PG-Gs and PG-EAs in vitro and in vivo. PGE(2)-G was rapidly hydrolyzed in rat plasma to generate PGE(2) (t(1/2) = 14 s) but was only slowly metabolized in human plasma (t(1/2) > 10 min). An intermediate extent of metabolism of PGE(2)-G was observed in human whole blood (t(1/2) approximately 7 min). The parent arachidonylglycerol, 2-AG, and the more stable regioisomer, 1-AG, also were much more rapidly metabolized in rat plasma compared with human plasma. PGE(2)-EA was not significantly hydrolyzed in plasma, undergoing slow dehydration/isomerization to PGB(2)-EA. Both PGE(2)-G and PGE(2)-EA were stable in canine, bovine, and human cerebrospinal fluid. Human 15-hydroxyprostaglandin dehydrogenase, the enzyme responsible for the initial step in PG inactivation in vivo, oxidized both PGE(2)-G and PGE(2)-EA less efficiently than the free acid. The sterically hindered glyceryl prostaglandin was the poorest substrate examined in the E series. Minimal 15-hydroxyprostaglandin dehydrogenase oxidation of PGF(2 alpha)-G was observed. PGE(2)-G and PGE(2)-EA pharmacokinetics were assessed in rats. PGE(2)-G was not detected in plasma 5 min following an intravenous dose of 2 mg/kg. However, PGE(2)-EA was detectable up to 2 h following an identical dose, displaying a large apparent volume of distribution and a half-life of over 6 min. The results suggest that endocannabinoid-derived PG-like compounds may be sufficiently stable in humans to exert actions systemically. Furthermore, these results suggest that the rat is not an adequate model for investigating the biological activities of 2-arachidonylglycerol or glyceryl prostaglandins in humans. (+info)
Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus.
(32/954)
In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity. (+info)