Recent progress in the neurotoxicology of natural drugs associated with dependence or addiction, their endogenous agonists and receptors.
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Nicotine in tobacco, tetrahydrocannabinol (delta 9-THC) in marijuana and morphine in opium are well known as drugs associated with dependence or addiction. Endogenous active substances that mimic the effects of the natural drugs and their respective receptors have been found in the mammalian central nervous system (CNS). Such active substances and receptors include acetylcholine (ACh) and the nicotinic ACh receptor (nAChR) for nicotine, anandamide and CB1 for delta 9-THC, and endomorphins (1 and 2) and the mu (OP3) opioid receptor for morphine, respectively. Considerable progress has been made in studies on neurotoxicity, in terms of the habituation, dependence and withdrawal phenomena associated with these drugs and with respect to correlations with endogenous active substances and their receptors. In this article we shall review recent findings related to the neurotoxicity of tobacco, marijuana and opium, and their toxic ingredients, nicotine, delta 9-THC and morphine in relation to their respective endogenous agents and receptors in the CNS. (+info)
Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development.
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Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation. (+info)
A role for N-arachidonylethanolamine (anandamide) as the mediator of sensory nerve-dependent Ca2+-induced relaxation.
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We tested the hypothesis that an endogenous cannabinoid (CB) receptor agonist, such as N-arachidonylethanolamine (anandamide), is the transmitter that mediates perivascular sensory nerve-dependent Ca2+-induced relaxation. Rat mesenteric branch arteries were studied using wire myography; relaxation was determined after inducing contraction with norepinephrine. Cumulative addition of Ca2+ caused dose-dependent relaxation (ED50 = 2.2 +/- 0.09 mM). The relaxation was inhibited by 10 mM TEA and 100 nM iberiotoxin, a blocker of large conductance Ca2+-activated K+ channels, but not by 5 microM glibenclamide, 1 mM 4-aminopyridine, or 30 nM apamin. Ca2+-induced relaxation was also blocked by the selective CB receptor antagonist SR141716A and was enhanced by pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc; 30 microM), an inhibitor of anandamide metabolism. Anandamide also caused dose-dependent relaxation (ED50 =.72 +/- 0.3 microM). The relaxation was not inhibited by endothelial denudation, 10 microM indomethacin, or 1 microM miconazole, but was blocked by 3 microM SR141716A, 10 mM TEA, precontraction with 100 mM K+, and 100 nM iberiotoxin, and was enhanced by treatment with 30 microM pefabloc. Mesenteric branch arteries were 200-fold more sensitive to the relaxing action of anandamide than arachidonic acid (ED50 = 160 +/- 7 microM). These data show that: 1) Ca2+ and anandamide cause hyperpolarization-mediated relaxation of mesenteric branch arteries, which is dependent on an iberiotoxin-sensitive Ca2+-activated K+ channel, 2) relaxation induced by both Ca2+ and anandamide is inhibited by CB receptor blockade, and 3) relaxation induced by anandamide is not dependent on its breakdown to arachidonic acid and subsequent metabolism. These findings support the hypothesis that anandamide, or a similar cannabinoid receptor agonist, mediates nerve-dependent Ca2+-induced relaxation in the rat. (+info)
Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists.
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1. The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, delta9-tetrahydrocannabinol (delta9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. 2. In rings of mesenteric artery HU 210 and delta9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03-30 microM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. 3. In endothelium-intact segments, HU 210, delta9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and delta9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. 4. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+ -signalling or cytochrome P450 activity. 5. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF. (+info)
Fatty-acid amide hydrolase is expressed in the mouse uterus and embryo during the periimplantation period.
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Arachidonoylethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors. We demonstrated previously that the periimplantation mouse uterus has high levels of anandamide and can synthesize and hydrolyse anandamide. In the present investigation, we examined the expression of the recently identified fatty-acid amide hydrolase (FAAH) gene, which is involved in hydrolyzing anandamide to arachidonic acid and ethanolamine, in the periimplantation mouse embryo and uterus. As previously reported, Northern blot hybridization detected a transcript of approximately 2.5 kilobases of FAAH mRNA in whole uterine poly(A)+ RNA samples. The levels of this mRNA were higher in the liver and brain than in the uterus. In the uterus, higher accumulation of FAAH mRNA occurred on Days 1-4 followed by declines on later days (Days 5-8) of pregnancy. In situ hybridization detected this mRNA primarily in uterine luminal and glandular epithelial cells on Days 1-4 of pregnancy. With the progression of implantation (Days 5-8), accumulation of this mRNA was retained in the luminal and glandular epithelia. In addition, implanting blastocysts showed accumulation of this mRNA. FAAH mRNA accumulation was absent or minimal in the myometrium during this period. Western blotting detected an approximately 60-kDa protein in uterine membrane preparations. In preimplantation embryos, FAAH mRNA was present in one-cell and two-cell embryos but was absent in embryos at the eight-cell/morula stage. However, this mRNA was again detected in Day 4 blastocysts. The presence of FAAH mRNA in one- and two-cell embryos reflects accumulation of maternal message, while its presence in blastocysts reflects embryonic gene activation. Collectively, our present and previous results provide evidence that FAAH is expressed in the mouse uterus and embryo during early pregnancy to modulate local levels of anandamide that could be important for embryo development and implantation. (+info)
Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts.
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The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, delta9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1-1 microM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 microM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system. (+info)
Anandamide activates human platelets through a pathway independent of the arachidonate cascade.
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Anandamide (arachidonoylethanolamide, AnNH) is shown to activate human platelets, a process which was not inhibited by acetylsalicylic acid (aspirin). Unlike AnNH, hydroperoxides generated thereof by lipoxygenase activity, and the congener (13-hydroxy)linoleoylethanolamide, were unable to activate platelets, though they counteracted AnNH-mediated stimulation. On the other hand, palmitoylethanolamide neither activated human platelets nor blocked the AnNH effects. AnNH inactivation by human platelets was afforded by a high-affinity transporter, which was activated by nitric oxide-donors up to 225% of the control. The internalized AnNH could thus be hydrolyzed by a fatty acid amide hydrolase (FAAH), characterized here for the first time. (+info)
Structural determinants for recognition and translocation by the anandamide transporter.
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The biological actions of anandamide (arachidonylethanolamide), an endogenous cannabinoid lipid, are terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na+-independent transporter that is selectively inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). In the present study, we examined the structural determinants governing recognition and translocation of substrates by the anandamide transporter constitutively expressed in a human astrocytoma cell line. Competition experiments with a select group of analogs suggest that substrate recognition by the transporter is favored by a polar nonionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it may serve as hydrogen acceptor. Thus, 2-arachidonylglycerol, a putative endogenous cannabinoid ester, also may serve as a substrate for the transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that no fewer than four cis nonconjugated double bonds are required for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation. These results outline the general structural requisites for anandamide transport and may assist in the development of selective inhibitors with potential clinical applications. (+info)