Role of endocannabinoids and endovanilloids in Ca signalling

Endocannabinoids and endovanilloids are, by definition, endogenous agonists at cannabinoid CB₁ or CB₂ receptors and transient receptor potential vanilloid-type-1 (TRPV1) channels, respectively. Due to the several ways through which cannabinoid receptors influence cytosolic Ca²⁺ concentrations, and to the fact that TRPV1 activation leads to the gating of cations, including Ca²⁺, both endocannabinoids and endovanilloids, taken separately, can strongly influence Ca²⁺ signalling. Moreover, CB₁/CB₂ receptors and TRPV1 channels are often expressed in the same or neighbouring cells, and this can lead to cross-talk between the two receptor types, which is further enriched by the fact that some endocannabinoids, like anandamide and N-arachidonoyldopamine, also activate TRPV1 channels. Finally, both endocannabinoids and endovanilloids also interact with non-cannabinoid, non-TRPV1 receptors and channels, and, although the full physiological relevance of such interactions is yet to be established, the “promiscuity” of these lipophilic molecules can increase even further the potential ways through which they affect Ca²⁺ signalling. Here we discuss the effects of endocannabinoids and endovanilloids on cytosolic Ca²⁺ concentrations and their potential biological consequences.     

Ligand-receptor signaling with endocannabinoids in preimplantation embryo development and implantation

Although adverse effects of cannabinoids on pregnancy have been indicated for many years, the mechanisms by which they exert their actions were not clearly understood. Only recently, molecular and biochemical approaches have led to the identification of two types of cannabinoid receptors, brain-type receptors (CB1-R) and spleen-type receptors (CB2-R), which mediate cannabinoid effects. These findings were followed by the discovery of endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG). The natural cannabinoids and endocannabinoids exert their effects via cannabinoid receptors and share similar pharmacological and physiological properties. Recent demonstration of expression of functional CB1-R in the preimplantation embryo and synthesis of anandamide in the pregnant uterus of mice suggests that cannabinoid ligand-receptor signaling is operative in the regulation of preimplantation embryo development and implantation. This review describes recent observations and their significance in embryo-uterine interactions during implantation and future research directions in this emerging area of interest.     

Restricted vs. unrestricted wheel running in mice: Effects on brain,behavior and endocannabinoids

Beneficial effects of voluntary wheel running on hippocampal neurogenesis, morphology and hippocampal-dependent behavior have widely been studied in rodents, but also serious side effects and similarities to stereotypy have been reported. Some mouse strains run excessively when equipped with running wheels, complicating the comparability to human exercise regimes. Here, we investigated how exercise restriction to 6 h/day affects hippocampal morphology and metabolism, stereotypic and basal behaviors, as well as the endocannabinoid system in wheel running C57BL/6 mice; the strain most commonly used for behavioral analyses and psychiatric disease models. Restricted and unrestricted wheel running had similar effects on immature hippocampal neuron numbers, thermoregulatory nest building and basal home-cage behaviors. Surprisingly, hippocampal gray matter volume, assessed with magnetic resonance (MR) imaging at 9.4 Tesla, was only increased in unrestricted but not in restricted runners. Moreover, unrestricted runners showed less stereotypic behavior than restricted runners did. However, after blockage of running wheels for 24 h stereotypic behavior also increased in unrestricted runners, arguing against a long-term effect of wheel running on stereotypic behavior. Stereotypic behaviors correlated with frontal glutamate and glucose levels assessed by ¹H-MR spectroscopy. While acute running increased plasma levels of the endocannabinoid anandamide in former studies in mice and humans, we found an inverse correlation of anandamide with the daily running distance after long-term running. In conclusion, although there are some diverging effects of restricted and unrestricted running on brain and behavior, restricted running does not per se seem to be a better animal model for aerobic exercise in mice.     

Organophosphate-sensitive lipases modulate brain lysophospholipids, ether lipids and endocannabinoids

Lipases play key roles in nearly all cells and organisms. Potent and selective inhibitors help to elucidate their physiological functions and associated metabolic pathways. Organophosphorus (OP) compounds are best known for their anticholinesterase properties but selectivity for lipases and other targets can also be achieved through structural optimization. This review considers several lipid systems in brain modulated by highly OP-sensitive lipases. Neuropathy target esterase (NTE) hydrolyzes lysophosphatidylcholine (lysoPC) as a preferred substrate. Gene deletion of NTE in mice is embryo lethal and the heterozygotes are hyperactive. NTE is very sensitive in vitro and in vivo to direct-acting OP delayed neurotoxicants and the related NTE-related esterase (NTE-R) is also inhibited in vivo. KIAA1363 hydrolyzes acetyl monoalkylglycerol ether (AcMAGE) of the platelet-activating factor (PAF) de novo biosynthetic pathway and is a marker of cancer cell invasiveness. It is also a detoxifying enzyme that hydrolyzes chlorpyrifos oxon (CPO) and some other potent insecticide metabolites. Monoacylglycerol lipase and fatty acid amide hydrolase regulate endocannabinoid levels with roles in motility, pain and memory. Inhibition of these enzymes in mice by OPs, such as isopropyl dodecylfluorophosphonate (IDFP), leads to dramatic elevation of brain endocannabinoids and distinct cannabinoid-dependent behavior. Hormone-sensitive lipase that hydrolyzes cholesteryl esters and diacylglycerols is a newly recognized in vivo CPO- and IDFP-target in brain. The OP chemotype can therefore be used in proteomic and metabolomic studies to further elucidate the biological function and toxicological significance of lipases in lipid metabolism. Only the first steps have been taken to achieve appropriate selective action for OP therapeutic agents.     

Role of early edema in the development of regional seizure-related brain damage

Kainic acid-induced seizures produced early (2 hr) generalized edema and later (24 and 48 hr) necrotic edema in temporal cortex and hippocampus as measured by specific gravity changes. Mannitol given during the seizure partially protected against the early edema and prevented the necrotic edema indicating early edema may play a role in later brain damage. However, H₂O intoxication, causing much greater generalized edema than the kainic acidinduced seizures, caused no necrotic edema in temporal cortex or hippocampus at 48 hr. Thus it appears that mannitol protection against kainic acid-induced brain damage may be by a mechanism in addition to dehydration.Special Issue dedicated to Dr. O. H. Lowry.     

Role of Slit proteins in the vertebrate brain.

Diffusible chemorepellents play a major role in guiding developing axons towards their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon's response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered.     

Vitamins and brain development.

Effects of deficiency of vitamins on early development of brain have been reviewed. Unusual developmental problems in neurogenesis specific for the brain and impairment of its functional capacities due to vitamin deficiency have been discussed. The species-specific "critical periods" in development of various systems have been mentioned. Indices such as reflex activity, locomotion, special senses, cognition and adaptive behavior were used for assessing brain maturation in experimental models and humans. Significant examples include brain anomalies in humans and other mammals caused by retinoid excess or deficit; increase in calbindin D28K, a vitamin D dependent calcium-binding protein during postnatal period in rat; hydrocephalus and exencephaly in prenatal rats and subarachnoidal or intracerebral hemorrhage in infants caused by vitamin E deficiency. Peripheral neuropathic lesions leading to infantile beriberi is caused by thiamine deficiency. Impaired growth in retinal layers leading to delay in maturation of electroretinogram and depth-perception in postnatal rats occur due to pyridoxine deficiency. Infants of severely vitamin B12 deficient mothers show abnormalities in behavior involving basal ganglia and pyramidal tract. Folic acid deficiency results in delayed maturation of the basic electroencepalographic patterns. In addition, vitamin-interactions leading to developmental errors have been pointed out. Vitamin B6 deficiency impairs vitamin B12 absorption and biotin deficiency may be aggravated by pantothenic acid deficiency. Vitamin C deficiency resulting in impaired metabolism may produce symptoms of deficiency of folic acid. Another characteristic examples is that iron absorption from dietary sources is dependent on ascorbic acid.     

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.     

Oxidative metabolism of endocannabinoids

There is increasing evidence that endocannabinoids play roles in a number of physiological and pathological processes ranging from the regulation of food intake to the inhibition of cancer cell proliferation. Consequently, multiple investigations into endocannabinoid metabolic disposition have been initiated. Such studies have begun to shed light on the mechanisms that regulate the endogenous cannabinoid system. In addition, they have identified a number of novel, endocannabinoid-derived lipids. In the future, these studies may form the foundation of efforts designed to subtly manipulate endocannabinoid tone in vivo to achieve therapeutic benefits without the profound side-effects observed with synthetic cannabinoid treatment.In addition to the well-studied hydrolytic mode of endocannabinoid metabolism, accumulating data suggest that these lipids are also susceptible to oxidative metabolism by a number of fatty acid oxygenases. These include the cyclooxygenases, lipoxygenases, and cytochrome P450s known to be involved in eicosanoid production from arachidonic acid. The available evidence concerning endocannabinoid oxidation is reviewed and the potential biological significance of this mode of metabolism is considered.     

Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.     

Modularity in vertebrate brain development and evolution.

Embryonic modularity and functional modularity are two principles of brain organization. Embryonic modules are histogenetic fields that are specified by position-dependent expression of patterning genes. Within each embryonic module, secondary and higher-level pattern formation takes places during development, finally giving rise to brain nuclei and cortical layers. Defined subsets of these structures become connected by fiber tracts to form the information-processing neural circuits, which represent the functional modules of the brain. We review evidence that a group of cell adhesion molecules, the cadherins, provides an adhesive code for both types of modularity, based on a preferentially homotypic binding mechanism. Embryonic modularity is transformed into functional modularity, in part by translating early-generated positional information into an array of adhesive cues, which regulate the binding of functional neural structures distributed across the embryonic modules. Brain modularity may provide a basis for adaptability in evolution.     

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.     

内源性大麻素对心血管系统的作用

在心肌组织、血管平滑肌细胞、内皮细胞和血管壁周围的神经纤维末梢以及血液中某些细胞存在内源性大麻素和相应的大麻素（CB）受体。在不同的动物模型和器官,内源性大麻素发挥调节血压和扩张血管等效应。内源性大麻素还在减少休克和心肌梗死所致循环和心脏损伤方面发挥重要作用,在心肌预适应中亦发挥关键作用。目前对内源性大麻素在心血管系统中作用的研究还处于起步阶段,本文对内源性大麻素系统在心血管系统中的来源和分布,对血管和心脏的作用及其机制方面的研究进展作简要介绍。     

Role of the brain in post-diapause adult development in the swallowtail, Papilio xuthus

The role of the brain in adult development was examined by brain removal in unchilled and chilled diapausing pupae of Papilio xuthus. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although photoperiod had little effect on diapause development. The brain played a role of shortening the pupal duration and synchronizing adult emergence in both unchilled and chilled individuals, although it was not essential for post-diapause adult development. The stimulus of low temperature was recorded even in the absence of the brain, because chilling shortened the pupal duration in brainless individuals. The brain showed activity which affected subsequent adult development in chilled pupae within one day after chilling in males. This period was less limited in females.