Analysis of cannabinoid receptor binding and mRNA expression and endogenous cannabinoid contents in the developing rat brain during late gestation and early postnatal period.

Cannabinoid CB(1) receptors emerge early in the rat brain during prenatal development, supporting their potential participation in events related to neural development. In the present investigation, we completed earlier studies, analyzing CB(1) receptor binding and mRNA expression by using autoradiography and in situ hybridization, respectively, in the brain of rat fetuses at gestational day (GD) 21 and of newborns at postnatal days (PND) 1 and 5, in comparison with the adult brain. These analyses were paralleled by quantitation of levels of anandamide and its precursor, N-arachidonoyl-phosphatidylethanolamine (NAPE), and of 2-arachidonoyl-glycerol (2-AG), carried out by using gas chromatography / mass spectrometry of the tri-methyl-sylyl-ether derivatives. As expected, CB(1) receptor binding was detected at GD21 in a variety of brain structures. In most of them, such as the hippocampus, cerebral cortex, cerebellum, basal ganglia, and limbic nuclei, there were no marked differences in the density of CB(1) receptors in animals at GD21 as compared to early newborns (PND1 and 5), although it markedly increased in these regions in adulthood. However, with the exception of the cerebellum and, in part, the caudate-putamen, the pattern observed for binding in these regions was clearly different from that observed for mRNA expression of the CB(1) receptor, which currently exhibited the highest levels at PND1 and the lowest in the adult brain. This was also seen in the basolateral amygdaloid nucleus, ventromedial hypothalamic nucleus, medial habenula, and other structures. In the caudate-putamen and, particularly, in the cerebellum, mRNA expression was higher in the adult brain as compared with other ages. As previously reported, specific binding for CB(1) receptors was also detected at GD21 in white matter areas, such as the corpus callosum, anterior commissure, fornix, fimbria, stria medullaris, stria terminalis, and fasciculus retroflexum. With the exception of the anterior commissure and the fimbria, specific binding progressively decreased at PND1 and PND5 until disappearing in the adult brain. In the fimbria, the highest values of binding were seen at PND1, but binding also completely disappeared in the adult brain, whereas in the anterior commissure, specific binding at PND1 and PND5 was lesser than that observed at GD21 and, particularly, in adulthood. CB(1) receptor mRNA expression was not detected in these white matter areas, thus dismissing the possible presence of these receptors in glial cells rather than in neuronal axons. However, mRNA expression was detected in the brainstem, an area also rich in white matter, and it mostly correlated with receptor binding, exhibiting a progressive decrease from GD21 up to adulthood. CB(1) receptor mRNA expression was also detected at GD21 in atypical areas where binding was not detected. These areas are proliferative regions, such as the subventricular zones of the neocortex, striatum, and nucleus accumbens. This atypical location only persisted at PND1 and PND5 in the striatal subventricular zone, but disappeared in the adult brain. We also found measurable levels of different endogenous cannabinoids in the developing brain. High levels of 2-AG, comparable to those found in the adult brain, were measured at GD21, whereas significantly lower levels were measured for anandamide and NAPE at this fetal age compared with the levels found in the adult brain. Levels of anandamide and NAPE increased during the early postnatal period until reaching the maximum in the adult brain. By contrast, 2-AG levels peaked at PND1, with values approximately twofold higher than those found at the other ages. In summary, all these data demonstrate that the endogenous cannabinoid system, constituted by endogenous ligands and receptor signaling pathways, is present in the developing brain, which suggests a possible specific role of this system in key processes of neural development. (c) 1999 Wiley-Liss, Inc.

Bioactive long chain N-acylethanolamines in five species of edible bivalve molluscs. Possible implications for mollusc physiology and sea food industry.

Several long chain N-acylethanolamines, including the proposed endogenous ligands of cannabinoid receptors, anandamide (N-arachidonoylethanolamine, C20:4 NAE) and N-palmitoylethanolamine (C16:0 NAE), as well as some of their putative biosynthetic precursors, the N-acyl-phosphatidylethanolamines, were found in lipid extracts of five species of bivalve molluscs, including Mytilus galloprovincialis, commonly used as sea food. The amounts of these metabolites, the most abundant being C16:0 NAE and N-stearoylethanolamine, appeared to increase considerably when mussels were extracted 24h post-mortem, but were not significantly affected by boiling the tissue prior to extraction. In particulate fractions of homogenates from Mytilus, where the existence of a highly selective cannabinoid receptor with an immunomodulatory function has been previously described, an enzymatic activity capable of catalyzing the hydrolysis of C20:4 NAE amide bond, and displaying similar pH dependency and inhibitor sensitivity profiles as the recently characterized 'fatty acid amide hydrolase' was found. The enzyme Km and Vmax for C20:4 NAE were 29.6 microM and 73 pmol/mg protein/min, respectively. These findings support the hypothesis that C20:4 NAE, never reported before in the phylum Mollusca, may be a mollusc physiological mediator, and suggest that edible bivalves may be a dietary, albeit limited, source of C16:0 NAE, whose anti-inflammatory properties, when administered orally in amounts higher than those reported here, have been previously reported.

The sleep inducing factor oleamide is produced by mouse neuroblastoma cells.

Cis-9,10-octadecenoamide (oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The enzyme catalyzing the hydrolysis of the amide bond of oleamide as well as of anandamide, the putative endogenous ligand of cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named fatty acid amide hydrolase (FAAH). The enzymatic synthesis of oleamide from oleic acid and ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of oleamide, necessary in order to postulate for this compound a role as a neuromodulator. Here we show for the first time that oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of oleic acid and ammonia. A lipid metabolite was extracted and purified from mouse neuroblastoma N18TG2 cells through a sequence of chromatographic steps and characterized as oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic oleamide, was 55.0+/-09.5 pmols/10(7) cells, compared to less than 0.7 pmol/10(7) cells for anandamide in the same cells. When N18TG2 cells were prelabeled with [14C]oleic acid and the lipids extracted and purified, a radioactive component with the same chromatographic behavior as oleamide was found whose levels: (1) were not significantly influenced by stimulation with ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF); (3) appeared to correlate with [14C]oleic acid incorporation into phospholipids but not with free [14C]oleic acid levels. N18TG2 cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of oleamide from oleic acid and ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and methylarachidonoylfluorophosphonate, as well as by an excess of anandamide, and by PMSF at the same concentration which increased oleamide formation in intact cells. These data suggest that a FAAH-like enzyme working "in reverse" may be responsible for the formation of oleamide in cell-free preparations but not in whole cells.

Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells.

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells.

Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role as peripheral mediators with multiple actions on blood cell function.

Treatment of chronic constipation by a bulk-forming laxative (Fibrolax).

Seventy-five patients affected by chronic constipation were treated for 4 weeks with an Ispaghula Husk preparation (Fibrolax), a bulk-forming laxative. Frequency, stool consistency, abdominal pain and signs of venous stasis improved after treatment. No important side-effect was recorded. Cholesterol, HDL-cholesterol and triglycerides did not show significant changes.