Tag: Cannabinoids

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

Satoshi Morimoto 1 , Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama and Futoshi Taura 

2007 The Journal of Biological Chemistry, 282, 20739-20751.

DOI: 10.1074/jbc.M700133200

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Δ1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nucleases released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

Abstract

GPR3 and GPR6, novel molecular targets for cannabidiol

Laun AS, Song ZH. 

Biochem Biophys Res Commun. 2017 Aug 12;490(1):17-21. 

doi: 10.1016/j.bbrc.2017.05.165

GPR3 and GPR6 are members of a family of constitutively active, Gs protein-coupled receptors. Previously, it has been reported that GPR3 is involved in Alzheimer’s disease whereas GPR6 plays potential roles in Parkinson’s disease. GPR3 and GPR6 are considered orphan receptors because there are no confirmed endogenous agonists for them. However, GPR3 and GPR6 are phylogenetically related to the cannabinoid receptors. In this study, the activities of endocannabinoids and phytocannabinoids were tested on GPR3 and GPR6 using a β-arrestin2 recruitment assay. Among the variety of cannabinoids tested, cannabidiol (CBD), the major non-psychoactive component of marijuana, significantly reduced β-arrestin2 recruitment to both GPR3 and GPR6. In addition, the inhibitory effects of CBD on β-arrestin2 recruitment were concentration-dependent for both GPR3 and GPR6, with a higher potency for GPR6. These data show that CBD acts as an inverse agonist at both GPR3 and GPR6 receptors. These results demonstrate for the first time that both GPR3 and GPR6 are novel molecular targets for CBD. Our discovery that CBD acts as a novel inverse agonist on both GPR3 and GPR6 indicates that some of the potential therapeutic effects of CBD (e.g. treatment of Alzheimer’s disease and Parkinson’s disease) may be mediated through these important receptors.

Abstract

Gene duplication and divergence affecting drug content in Cannabis sativa

George D. Weiblen, Jonathan P. Wenger, Kathleen J. Craft, Mahmoud A. ElSohly, Zlatko Mehmedic, Erin L. Treiber, M. David Marks

New Phytologist 17 July 2015

DOI: 10.1111/nph.13562

Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

Abstract

Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L.

Ross, S. A., ElSohly, M. A., Sultana, G. N. N., Mehmedic, Z., Hossain, C. F., & Chandra, S. 

Phytochemical Analysis, 16(1), 45–48. (2005). 

doi: 10.1002/pca.809

Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavonol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-O-sophoroside by spectroscopic methods including high-field two-dimensional NMR experiments. The characterisation of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannabinoids and reported data. The identified cannabinoids were ∆9 – tetrahydrocannabiorcol, cannabidivarin, cannabicitran, ∆9 -tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, ∆9 -tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol , 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-Oethylcannabitriol.

Abstract

First direct evidence for the mechanism of .DELTA.1-tetrahydrocannabinolic acid biosynthesis

Taura, F., Morimoto, S., Shoyama, Y., & Mechoulam, R. 

Journal of the American Chemical Society, 117(38), 9766–9767 (1995). 

doi: 10.1021/ja00143a024

Numerous plausible hypotheses have been advanced regarding the biogenesis of A’-tetrahydrocannabinol (AI-THC, la), the psychoactive constituent of marihuana (Cannabis satiua L.);I however, they all lack experimental support. Thus all hypothetical biogenetic schemes assume that Al tetrahydrocannabinolic acid (AI-THCA, lb), the precursor of AI-THC, is formed by cyclization from cannabidiolic acid (2).2 We now present experimental evidence that establishes that lb is actually formed from cannabigerolic acid (3b) through oxidocyclization by a new enzyme, AI-THCA synthase, and that 2, a plausible intermediate, is not a substrate for this enzymatic reaction.

Abstract

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes

Oier Aizpurua-Olaizola, Umut Soydaner, Ekin Öztürk, Daniele Schibano, Yilmaz Simsir, Patricia Navarro, Nestor Etxebarria, and Aresatz Usobiaga

Journal of Natural Products (2016)

DOI: 10.1021/acs.jnatprod.5b00949

The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

Abstract

Evidence that the plant cannabinoid Δ9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist.

Thomas, A., Stevenson, L. A., Wease, K. N., Price, M. R., Baillie, G., Ross, R. A., & Pertwee, R. G.

British Journal of Pharmacology, 146(7), 917–926. (2005). 

doi: 10.1038/sj.bjp.0706414

Cannabis sativa is the natural source of a set of at least 66 oxygen-containing aromatic hydrocarbon compounds that are known collectively as phytocannabinoids (reviewed in ElSohly, 2002). This study focused on a little-investigated phytocannabinoid, the n-propyl analogue of D9 -tetrahydrocannabinol (THC) (Figure 1), which was first detected in cannabis by Gill et al. (1970) and named D9 -tetrahydrocannabivarin (THCV) by Merkus (1971). The initial objective of this research was to establish whether THCV can activate or block cannabinoid CB1 or CB2 receptors.

Introduction

Evaluation of prevalent phytocannabinoids in the acetic acid model of visceral nociception.

Booker, L., Naidu, P. S., Razdan, R. K., Mahadevan, A., & Lichtman, A. H. 

Drug and Alcohol Dependence, 105(1-2), 42–47(2009). 

doi: 10.1016/j.drugalcdep.2009.06.009

Considerable preclinical research has demonstrated the efficacy of 9-tetrahydrocannabinol (9-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of 9-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, 9-THC and CBN bound to the CB1 receptor and produced antinociceptive effects. The CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB1 receptor with similar affinity as 9-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of 9-THC when both drugs were given in combination. Importantly, the antinociceptive effects of 9-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of 9-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of 9-THC.

Engineering yeasts as platform organisms for cannabinoid biosynthesis.

Zirpel, B., Degenhardt, F., Martin, C., Kayser, O., & Stehle, F. 

Journal of Biotechnology, 259, 204–212 (2017).

doi: 10.1016/j.jbiotec.2017.07.008

Δ 9 -tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa L. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme’s coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and 3 geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.

Abstract

Endogenous cannabinoids and appetite

Tim C. Kirkham and Claire M. Williams

Nutrition Research Reviews (2001), 14, 65–86 

DOI: 10.1079/NRR200118

Since pre-history, Cannabis sativa has been exploited for its potent and manifold pharmacological actions. Amongst the most renowned of these actions is a tendency to provoke ravenous eating. The characterization of the psychoactive principals in cannabis (exogenous cannabinoids) and, more recently, the discovery of specific brain cannabinoid receptors and their endogenous ligands (endocannabinoids) has stimulated research into the physiological roles of endocannabinoid systems. In this review, we critically discuss evidence from the literature that describe studies on animals and human subjects to support endocannabinoid involvement in the control of appetite. We describe the hyperphagic actions of the exogenous cannabinoid, 9-tetrahydrocannabinol, and the endogenous CB1 ligands, anandamide and 2-arachidonylglycerol, and present evidence to support a specific role of endocannabinoid systems in appetitive processes related to the incentive and reward properties of food. A case is made for more comprehensive and systematic analyses of cannabinoid actions on eating, in the anticipation of improved therapies for disorders of appetite and body weight, and a better understanding of the biopsychological processes underlying hunger.

Abstract