Origins of Cannabis sativa in Ireland and the Concept of Celtic Hemp: An Interdisciplinary Review.

Introduction: Ireland's agriculture has been shaped by Celts, Romano-British Christians, Norse-Vikings, Anglo-Normans, and subsequent migrants. Who introduced hemp (Cannabis sativa) to Hibernia? We addressed this question using historical linguistics, fossil pollen studies (FPSs), archaeological data, and written records. Methods: Data gathering utilized digital resources coupled with citation tracking. Linguistic methods separated cognates (words with shared etymological origins) from loanwords (borrowed from other languages). Cannabis pollen in FPSs was identified using the "ecological proxy" method. Archaeological reports were ranked on a "robustness" scale. Results: Words for "hemp" in Celtic languages are loanwords, not cognates. The Irish word cnáib is first attested in texts written 1060 and 1127-1134 CE. Old Breton coarcholion, corrected to coarch, is attested in a text from the 9th century. Pollen consistent with cultivated Cannabis appears in the Middle Ages, ca. 700 CE, at sites in the vicinity of monasteries. Archaeological finds (hemp seeds and fiber) date to later Norse-Viking and Anglo-Norman sites. Discussion: People of the Hallstatt Culture in Central Europe have long been considered speakers of the "Proto-Celtic" language. The lack of "hemp" cognates means a Proto-Celtic word cannot be reconstructed, which implies that Hallstatt people (with robust archaeological evidence of hemp) did not speak Proto-Celtic. Cnáib is absent in Old Irish glossaries, epics, and mythologies (600-900 CE). FPS data suggest that the onset of hemp cultivation correlated-chronologically and spatially-with the founding of Romano-British monasteries. Irish cnáib was likely borrowed from Clerical Latin canapis or canabus.

A classification of endangered high-THC cannabis (Cannabis sativa subsp. indica) domesticates and their wild relatives.

Two kinds of drug-type Cannabis gained layman's terms in the 1980s. "Sativa" had origins in South Asia (India), with early historical dissemination to Southeast Asia, Africa, and the Americas. "Indica" had origins in Central Asia (Afghanistan, Pakistan, Turkestan). We have assigned unambiguous taxonomic names to these varieties, after examining morphological characters in 1100 herbarium specimens, and analyzing phytochemical and genetic data from the literature in a meta-analysis. "Sativa" and "Indica" are recognized as C. sativa subsp. indica var. indica and C. sativa subsp. indica var. afghanica, respectively. Their wild-growing relatives are C. sativa subsp. indica var. himalayensis (in South Asia), and C. sativa subsp. indica var. asperrima (in Central Asia). Natural selection initiated divergence, driven by climatic conditions in South and Central Asia. Subsequent domestication drove further phytochemical divergence. South and Central Asian domesticates can be distinguished by tetrahydrocannabinol and cannabidiol content (THC/CBD ratios, ≥7 or <7, respectively), terpenoid profiles (absence or presence of sesquiterpene alcohols), and a suite of morphological characters. The two domesticates have undergone widespread introgressive hybridization in the past 50 years. This has obliterated differences between hybridized "Sativa" and "Indica" currently available. "Strains" alleged to represent "Sativa" and "Indica" are usually based on THC/CBD ratios of plants with undocumented hybrid backgrounds (with so-called "Indicas" often delimited simply on possession of more CBD than "Sativas"). The classification presented here circumscribes and names four taxa of Cannabis that represent critically endangered reservoirs of germplasm from which modern cannabinoid strains originated, and which are in urgent need of conservation.

Experimental Endozoochory of Cannabis sativa Achenes.

The mechanism by which Cannabis sativa dispersed from its center of origin remains an open question. The literature provides many hypotheses, which we review for the first time, but experiments are few. Darwin was interested in zoochory - the transport of plants by animals. He demonstrated endozoochory (transport of seeds via animal digestive systems) of C. sativa achenes (seeds) by carrier pigeons, but he did not quantify achene survival rates. We assessed mammalian endozoochory in a triplicate experiment: feeding C. sativa achenes into a simulated gastrointestinal system, a dog, and a human. The in vitro system subjected achenes to sequential digestive enzymes. Achenes were planted in potting soil and monitored for emergence under growroom conditions. The in vivo experiments added achenes to a normal morning meal (dog food or granola). Feces were collected for daily instillation into an outdoor garden and monitored for seedling emergence for 16 days. Control achenes were planted directly into soil without ingestion. In the in vitro study, 34.7% of the digested achenes emerged as seedlings. The in vivo emergence rates were 10.3, 1.3, and 76.0% for the dog, human, and control conditions. The three groups differed significantly (χ2 = 1,264.93, p < 0.0001). Achene survival was greatest under in vitro conditions, which lacked a mastication step, compared to dog (minimal chewing) and human (maximal chewing) conditions. Although C. sativa lacks evolutionary traits for classic endozoochory (i.e., a fleshy fruit), it seems well adapted to this manner of seed dispersal.

Cannabis Systematics at the Levels of Family, Genus, and Species.

New concepts are reviewed in Cannabis systematics, including phylogenetics and nomenclature. The family Cannabaceae now includes Cannabis, Humulus, and eight genera formerly in the Celtidaceae. Grouping Cannabis, Humulus, and Celtis actually goes back 250 years. Print fossil of the extinct genus Dorofeevia (=Humularia) reveals that Cannabis lost a sibling perhaps 20 million years ago (mya). Cannabis print fossils are rare (n=3 worldwide), making it difficult to determine when and where she evolved. A molecular clock analysis with chloroplast DNA (cpDNA) suggests Cannabis and Humulus diverged 27.8 mya. Microfossil (fossil pollen) data point to a center of origin in the northeastern Tibetan Plateau. Fossil pollen indicates that Cannabis dispersed to Europe by 1.8-1.2 mya. Mapping pollen distribution over time suggests that European Cannabis went through repeated genetic bottlenecks, when the population shrank during range contractions. Genetic drift in this population likely initiated allopatric differences between European Cannabis sativa (cannabidiol [CBD]>Δ9-tetrahydrocannabinol [THC]) and Asian Cannabis indica (THC>CBD). DNA barcode analysis supports the separation of these taxa at a subspecies level, and recognizing the formal nomenclature of C. sativa subsp. sativa and C. sativa subsp. indica. Herbarium specimens reveal that field botanists during the 18th-20th centuries applied these names to their collections rather capriciously. This may have skewed taxonomic determinations by Vavilov and Schultes, ultimately giving rise to today's vernacular taxonomy of "Sativa" and "Indica," which totally misaligns with formal C. sativa and C. indica. Ubiquitous interbreeding and hybridization of "Sativa" and "Indica" has rendered their distinctions almost meaningless.

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals, birds, and amphibians.

PURPOSE: Previous studies have found non-CB1 non-CB2 G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians. MATERIALS AND METHODS: Antagonism of the stimulation of [35S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors. RESULTS: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [35S]GTPγS binding by WIN55212-2 was not blocked by the CB1 antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian. CONCLUSIONS: The portion of WIN55212-2-stimulated [35S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB1 receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.

Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two.

Introduction:Cannabis biosynthesizes Δ9-tetrahydrocannabinolic acid (THCA-A), which decarboxylates into Δ9-tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB1). Materials and Methods: Purity of certified reference standards were tested with high performance liquid chromatography (HPLC). Binding affinity of THCA-A and THC at human (h) CB1 and hCB2 was measured in competition binding assays, using transfected HEK cells and [3H]CP55,940. Efficacy at hCB1 and hCB2 was measured in a cyclic adenosine monophosphase (cAMP) assay, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor. Results: The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB1 and hCB2, equating to approximate Ki values of 3.1µM and 12.5µM, respectively. THC showed 62-fold greater affinity at hCB1 and 125-fold greater affinity at hCB2. In efficacy tests, THCA-A (10µM) slightly inhibited forskolin-stimulated cAMP at hCB1, suggestive of weak agonist activity, and no measurable efficacy at hCB2. Discussion: The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10µM approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact-from its inevitable decarboxylation into THC-and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB1 or CB2.

Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review.

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ(9) -tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ(9) -tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target.

Care and feeding of the endocannabinoid system: a systematic review of potential clinical interventions that upregulate the endocannabinoid system.

BACKGROUND: The "classic" endocannabinoid (eCB) system includes the cannabinoid receptors CB1 and CB2, the eCB ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and their metabolic enzymes. An emerging literature documents the "eCB deficiency syndrome" as an etiology in migraine, fibromyalgia, irritable bowel syndrome, psychological disorders, and other conditions. We performed a systematic review of clinical interventions that enhance the eCB system--ways to upregulate cannabinoid receptors, increase ligand synthesis, or inhibit ligand degradation. METHODOLOGY/PRINCIPAL FINDINGS: We searched PubMed for clinical trials, observational studies, and preclinical research. Data synthesis was qualitative. Exclusion criteria limited the results to 184 in vitro studies, 102 in vivo animal studies, and 36 human studies. Evidence indicates that several classes of pharmaceuticals upregulate the eCB system, including analgesics (acetaminophen, non-steroidal anti-inflammatory drugs, opioids, glucocorticoids), antidepressants, antipsychotics, anxiolytics, and anticonvulsants. Clinical interventions characterized as "complementary and alternative medicine" also upregulate the eCB system: massage and manipulation, acupuncture, dietary supplements, and herbal medicines. Lifestyle modification (diet, weight control, exercise, and the use of psychoactive substances--alcohol, tobacco, coffee, cannabis) also modulate the eCB system. CONCLUSIONS/SIGNIFICANCE: Few clinical trials have assessed interventions that upregulate the eCB system. Many preclinical studies point to other potential approaches; human trials are needed to explore these promising interventions.

Obesity, the endocannabinoid system, and bias arising from pharmaceutical sponsorship.

BACKGROUND: Previous research has shown that academic physicians conflicted by funding from the pharmaceutical industry have corrupted evidence based medicine and helped enlarge the market for drugs. Physicians made pharmaceutical-friendly statements, engaged in disease mongering, and signed biased review articles ghost-authored by corporate employees. This paper tested the hypothesis that bias affects review articles regarding rimonabant, an anti-obesity drug that blocks the central cannabinoid receptor. METHODS/PRINCIPAL FINDINGS: A MEDLINE search was performed for rimonabant review articles, limited to articles authored by USA physicians who served as consultants for the company that manufactures rimonabant. Extracted articles were examined for industry-friendly bias, identified by three methods: analysis with a validated instrument for monitoring bias in continuing medical education (CME); analysis for bias defined as statements that ran contrary to external evidence; and a tally of misrepresentations about the endocannabinoid system. Eight review articles were identified, but only three disclosed authors' financial conflicts of interest, despite easily accessible information to the contrary. The Takhar CME bias instrument demonstrated statistically significant bias in all the review articles. Biased statements that were nearly identical reappeared in the articles, including disease mongering, exaggerating rimonabant's efficacy and safety, lack of criticisms regarding rimonabant clinical trials, and speculations about surrogate markers stated as facts. Distinctive and identical misrepresentations regarding the endocannabinoid system also reappeared in articles by different authors. CONCLUSIONS: The findings are characteristic of bias that arises from financial conflicts of interest, and suggestive of ghostwriting by a common author. Resolutions for this scenario are proposed.

Expression of the endocannabinoid system in fibroblasts and myofascial tissues.

The endocannabinoid (eCB) system, like the better-known endorphin system, consists of cell membrane receptors, endogenous ligands and ligand-metabolizing enzymes. Two cannabinoid receptors are known: CB(1) is principally located in the nervous system, whereas CB(2) is primarily associated with the immune system. Two eCB ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are mimicked by cannabis plant compounds. The first purpose of this paper was to review the eCB system in detail, highlighting aspects of interest to bodyworkers, especially eCB modulation of pain and inflammation. Evidence suggests the eCB system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, expression of the eCB system in myofascial tissues has not been established. The second purpose of this paper was to investigate the eCB system in fibroblasts and other fascia-related cells. The investigation used a bioinformatics approach, obtaining microarray data via the GEO database (www.ncbi.nlm.nih.gov/geo/). GEO data mining revealed that fibroblasts, myofibroblasts, chondrocytes and synoviocytes expressed CB(1), CB(2) and eCB ligand-metabolizing enzymes. Fibroblast CB(1) levels nearly equalled levels expressed by adipocytes. CB(1) levels upregulated after exposure to inflammatory cytokines and equiaxial stretching of fibroblasts. The eCB system affects fibroblast remodeling through lipid rafts associated with focal adhesions and dampens cartilage destruction by decreasing fibroblast-secreted metalloproteinase enzymes. In conclusion, the eCB system helps shape biodynamic embryological development, diminishes nociception and pain, reduces inflammation in myofascial tissues and plays a role in fascial reorganization. Practitioners wield several tools that upregulate eCB activity, including myofascial manipulation, diet and lifestyle modifications, and pharmaceutical approaches.

The endocannabinoid system: an osteopathic perspective.

The present review provides an update on endocannabinoid basic science and clinical studies and proposes a new model to describe reciprocal interactions between somatic dysfunction and the endocannabinoid system. The endocannabinoid system consists of cannabinoid receptors, endogenous ligands, and ligand-metabolizing enzymes. The system exemplifies the osteopathic principle that the body possesses self-regulatory mechanisms that are self-healing in nature. Enhancing endocannabinoid activity has broad therapeutic potential, including the treatment of patients with somatic dysfunction, chronic pain, and neurodegenerative diseases as well as inflammatory conditions, bowel dysfunctions, and psychological disorders. Blockade of the endocannabinoid system with drugs such as rimonabant and taranabant may oppose self-healing mechanisms and elicit adverse effects. Osteopathic physicians wield several tools that can augment endocannabinoid activity, including lifestyle modifications, pharmaceutical approaches, and osteopathic manipulative treatment.

Cannabimimetic effects modulated by cholinergic compounds.

This report is based upon a clinical case series describing five patients who volitionally adultered cannabis with a variety of compounds that shared a common trait-cholinergic modulation. They included a nicotinic agonist, muscarinic antagonist and antiacetylcholinesterase compounds. Some of these compounds (e.g. tobacco) are known to exert pharmacokinetic effects upon cannabinoids (e.g. improved drug absorption). Contrarily, our patients claimed that the compounds altered pharmacodynamic 'cannabimimetic' effects. The case series was supported by forensic identification of adulterants and by use of a symptom causality algorithm. A survey of the gray literature and drug culture web sites indicated that the case series portended a larger social phenomenon. Furthermore, many clinical reports, animal behaviour studies and in vitro mechanistic studies substantiated our observations. In conclusion, we provide empirical data regarding a new trend in the drug culture-cholinergic modulation of cannabinoid effects-that presents new research directions.

Cannabinoid CB1 and CB2 receptor ligand specificity and the development of CB2-selective agonists.

Cannabinoids in current use such as nabilone activate both CB1 and CB2 receptors. Selective CB2 activation may provide some of the therapeutic effects of cannabinoids, such as their immuno-modulatory properties, without the psychoactive effects of CB1 activation. Therefore, cannabinoid CB2 receptors represent an attractive target for drug development. However, selective and potent CB2 agonists remain in development. CB1 and CB2 differ considerably in their amino acid sequence and tertiary structures. Therefore, clinical development of potent and selective CB2 agonists is probable. Mutational and ligand binding studies, functional mapping, and computer modelling have revealed key residues and domains in cannabinoid receptors that are involved in agonist and antagonist binding to CB1 and CB2. In addition, CB2 has undergone more rapid evolution, and results for ligand binding and efficacy cannot be automatically extrapolated from rat or mouse CB2 to human. Furthermore, loss of CB1 affinity is a crucial property for CB2-selective ligands, and although rat CB1 is 97% homologous with human CB1, critical differences do exist, with potential for further exploitation in drug design. In this paper we briefly review previous cannabinoid receptor models and mutation/binding studies. We also review binding affinity ratios with respect to CB1 and CB2. We then employ our own models to illustrate key cannabinoid receptor residues and binding subdomains that are involved in these differences in binding affinities and discuss how these might be exploited in the development of CB2 specific ligands. Published reports for species specific binding affinities for CB2 are scarce, and we argue that this needs to be corrected prior to the progression of CB2 agonists from pre-clinical to clinical research.

Tempo and mode in the endocannaboinoid system.

The best-known endocannabinoid ligands, anandamide and 2-AG, signal at least seven receptors and involve ten metabolic enzymes. Genes for the receptors and enzymes were examined for heterogeneities in tempo (relative rate of evolution, RRE) and mode (selection pressure, Ka/Ks) in six organisms with sequenced genomes. BLAST identified orthologs as reciprocal best hits, and nucleotide alignments were performed with ClustalX and MacClade. Two bioinformatics platforms, LiKaKs (a distance-based LWL85 model) and SNAP (a parsimony-based NG86 model) made pairwise comparisons of orthologs in murids (rat and mouse) and primates (human and macaque). Mean RRE of the 18 endocannabinoid genes was significantly greater in murids than primates, whereas mean Ka/Ks did not differ significantly. Next we used FUGE (tree-based maximum-likelihood model) to compute human lineage-specific Ka/Ks calculations for 18 genes, which ranged from 1.11 to 0.00, in rank order from highest to lowest: PTPN22, NAAA, TRPV1, TRPA1, NAPE-PLD, MAGL, PPARgamma, FAAH1, COX2, FAAH2, ABDH4, CB2, GPR55, DAGLbeta, PPARalpha, TRPV4, CB1, DAGLalpha; differences were significant (p < 0.0001). Rat and mouse presented different rank orders (e.g., GPR55 generated the greatest Ka/Ks ratio). The 18 genes were then tested for recent positive selection (within 10,000 yr) using an extended haplotype homozygosity analysis of SNP data from the HapMap database. Significant evidence (p < 0.05) of a positive "selective sweep" was exhibited by PTPN22, TRPV1, NAPE-PLD, and DAGLalpha. In conclusion, the endocannabinoid system is collectively under strong purifying selection, although some genes show evidence of adaptive evolution.

Coevolution between cannabinoid receptors and endocannabinoid ligands.

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.

Evolutionary origins of the endocannabinoid system.

Endocannabinoid system evolution was estimated by searching for functional orthologs in the genomes of twelve phylogenetically diverse organisms: Homo sapiens, Mus musculus, Takifugu rubripes, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, Plasmodium falciparum, Tetrahymena thermophila, Archaeoglobus fulgidus, and Mycobacterium tuberculosis. Sequences similar to human endocannabinoid exon sequences were derived from filtered BLAST searches, and subjected to phylogenetic testing with ClustalX and tree building programs. Monophyletic clades that agreed with broader phylogenetic evidence (i.e., gene trees displaying topographical congruence with species trees) were considered orthologs. The capacity of orthologs to function as endocannabinoid proteins was predicted with pattern profilers (Pfam, Prosite, TMHMM, and pSORT), and by examining queried sequences for amino acid motifs known to serve critical roles in endocannabinoid protein function (obtained from a database of site-directed mutagenesis studies). This novel transfer of functional information onto gene trees enabled us to better predict the functional origins of the endocannabinoid system. Within this limited number of twelve organisms, the endocannabinoid genes exhibited heterogeneous evolutionary trajectories, with functional orthologs limited to mammals (TRPV1 and GPR55), or vertebrates (CB2 and DAGLbeta), or chordates (MAGL and COX2), or animals (DAGLalpha and CB1-like receptors), or opisthokonta (animals and fungi, NAPE-PLD), or eukaryotes (FAAH). Our methods identified fewer orthologs than did automated annotation systems, such as HomoloGene. Phylogenetic profiles, nonorthologous gene displacement, functional convergence, and coevolution are discussed.

Cannabimimetic effects of osteopathic manipulative treatment.

Endogenous cannabinoids activate cannabinoid receptors in the brain and elicit mood-altering effects. Parallel effects (eg, anxiolysis, analgesia, sedation) may be elicited by osteopathic manipulative treatment (OMT), and previous research has shown that the endorphin system is not responsible for OMT's mood-altering effects. The authors investigate whether OMT generated cannabimimetic effects for 31 healthy subjects in a dual-blind, randomized controlled trial that measured changes in subjects' scores on the 67-item Drug Reaction Scale (DRS). Chemical ionization gas chromatography and mass spectrometry were also used to determine changes in serum levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), and oleylethanolamide (OEA). In subjects receiving OMT, posttreatment DRS scores increased significantly for the cannabimimetic descriptors good, high, hungry, light-headed, and stoned, with significant score decreases for the descriptors inhibited, sober, and uncomfortable. Mean posttreatment AEA levels (8.01 pmol/mL) increased 168% over pretreatment levels (2.99 pmol/mL), mean OEA levels decreased 27%, and no changes occurred in 2-AG levels in the group receiving OMT. Subjects in the sham manipulative treatment group recorded mixed DRS responses, with both increases and decreases in scores for cannabimimetic and noncannabimimetic descriptors and no changes in sera levels. When changes in serum AEA were correlated with changes in subjects' DRS scores, increased AEA correlated best with an increase for the descriptors cold and rational, and decreased sensations for the descriptors bad, paranoid, and warm. The authors propose that healing modalities popularly associated with changes in the endorphin system, such as OMT, may actually be mediated by the endocannabinoid system.