Tag: In Vitro

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways

O Gomez, A Sanchez‐Rodriguez, MQU Le, C Sanchez‐Caro, F Molina‐Holgado, E Molina‐Holgado

British Journal of Pharmacology (2011) 163 1520–1532

doi: 10.1111/j.1476-5381.2011.01414.x

The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocytedifferentiation , we exposed differentiating cultures of oligodendrocytes with cannabinoid CB1, CB2 and CB1/CB2 receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity.
Background & Purpose

The psychoactive plant cannabinoid, Δ9-tetrahydrocannabinol, is antagonized by Δ8- and Δ9-tetrahydrocannabivarin in mice in vivo.

RG Pertwee, A Thomas, LA Stevenson, RA Ross, SA Varvel, AH Lichtman, BR Martin and RK Razdan

British Journal of Pharmacology, 150(5), 586–594. (2009)

doi: 10.1038/sj.bjp.0707124

To follow up in vitro evidence that D9-tetrahydrocannabivarin extracted from cannabis (D9-THCV) is a CB1 receptor antagonist by establishing whether synthetic D9-tetrahydrocannabivarin (O-4394) and D8-tetrahydrocannabivarin (O-4395) behave as CB1 antagonists in vivo.
O-4394 and O-4395 were compared with eD9 -THCV as displacers of [3H]-CP55940 from specific CB1 binding sites on mouse brain membranes and as antagonists of CP55940 in [35S]GTPgS binding assays performed with mouse brain membranes and of R-( þ )-WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg1 (i.v.) D9-tetrahydrocannabinol in mice was then investigated.
Background & Purpose, Experimental Approach

The phytocannabinoid Δ9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum.

Y-L Ma, SE Weston, BJ Whalley and GJ Stephens

British Journal of Pharmacology, 154(1), 204–215. (2008)

doi: 10.1038/bjp.2008.57

The phytocannabinoid D9-tetrahydrocannabivarin (D9-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of D9-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS.
Effects of D9-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro.
Background & Purpose, Experimental Approach

The phytocannabinoid, Δ9-tetrahydrocannabivarin, can act through 5-HT1A receptors to produce anti-psychotic effects

Maria grazia Cascio, Erica Zamberletti, Pietro Marini, Roger G Pertwee

November 2014 British Journal of Pharmacology 172(5)

DOI: 10.1111/bph.13000

To address the questions of whether Δ9-tetrahydrocannabivarin (THCV) can (a) enhance activation of 5-HT1A receptors in vitro and (b) induce any apparent 5-HT1A receptor-mediated anti-psychotic effects in vivo.
In vitro studies investigated the effect of THCV on targeting by 8-hydroxy-2 (di-npropylamino)tetralin (8-OH-DPAT) of 5-HT1A receptors in membranes obtained from rat brainstem or human 5-HT1A CHO cells, using [35S]GTPγS and 8-[3H]-OH-DPAT binding assays. In vivo studies investigated whether THCV induces signs of 5-HT1A receptor-mediated antipsychotic effects in rats
Background & Purpose, Experimental Approach

Regulation of Cannabinoid CB1 Receptors in the Central Nervous System by Chronic Cannabinoids

Laura J. Sim-Selley 

Critical Reviews™ in Neurobiology Volume 15, 2003 Issue 2 30 pages

DOI: 10.1615/CritRevNeurobiol.v15.i2.10

Marijuana produces a number of characteristic behaviors in humans and animals, including memory impairment, antinociception, and locomotor and psychoactive effects. However, tolerance and dependence to cannabinoids develops after chronic use, as demonstrated both clinically and in animal models. The potential therapeutic benefits of certain cannabinoid-mediated effects, as well as the use of marijuana for its psychoactive properties, has raised interest in understanding the cellular adaptations produced by chronic administration of this class of drugs. The primary active constituent of marijuana, delta9-tetrahydrohydrocannabinol (THC), binds to specific G-protein-coupled receptors. The central nervous system (CNS) effects of THC are mediated by CB1 receptors, which couple primarily to inhibitory G-proteins. High levels of CB1 receptors are found in the basal ganglia, hippocampus, cortex, and cerebellum, consistent with the profile of behavioral effects. Studies over the past decade have determined that CB1 receptors undergo downregulation and desensitization following chronic administration of THC or synthetic cannabinoid agonists. In general, these adaptations are regionally widespread and of considerable magnitude, and are thought to contribute to tolerance to cannabinoid- mediated behavioral effects. Adaptation at the effector level has been more difficult to characterize, although it appears that alterations in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity may be particularly important in cannabinoid dependence. A striking characteristic of CB1 receptor adaptation is the region dependence of the magnitude and rate of development of downregulation and desensitization. These regional differences may provide interesting insights into the mechanisms of CB1 receptors receptor signaling in different brain regions. Moreover, region-specific adaptations in CB1 receptors following chronic cannabinoid administration may produce differential adaptations at the in vivo level.
Abstract

Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa l.

Zirpel, B., Stehle, F., & Kayser, O. 

Biotechnology Letters, 37(9), 1869–1875. (2015). 

doi: 10.1007/s10529-015-1853-x

Objective- The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of D9- tetrahydrocannabinolic acid (THCA) using whole cells.
Results- Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after 192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1 ) THCA could be produced by 10.5 gCDW l -1 before enzyme activity was lost.
Abstract

Production of Δ1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

Taura, F., Dono, E., Sirikantaramas, S., Yoshimura, K., Shoyama, Y., & Morimoto, S. 

Biochemical and Biophysical Research Communications, 361(3), 675–680 (2007)

doi: 10.1016/j.bbrc.2007.07.079

D1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of D1 -tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted 1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.
Abstract

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

Designing microorganisms for heterologous biosynthesis of cannabinoids

Carvalho, Â., Hansen, E. H., Kayser, O., Carlsen, S., & Stehle, F. 
FEMS Yeast Research, 17(4).(2017).
DOI: 10.1093/femsyr/fox037

During the last decade the use of medical Cannabis has expanded globally and legislation is getting more liberal in many countries, facilitating the research on cannabinoids. The unique interaction of cannabinoids with the human endocannabinoid system make these compounds an interesting target to be studied as a therapeutic agent for the treatment of several medical conditions. However, currently there are important limitations in the study, production and use of cannabinoids as pharmaceutical drugs. Besides the main constituent tetrahydrocannabinolic acid, the structurally related compound cannabidiol is of high interest as drug candidate. From the more than 100 known cannabinoids most of them are mostly unknown with regard to their pharmacological profile and in very low amounts extractable. Today, cannabinoids are isolated from the strictly regulated Cannabis plant and the supply of compounds with sufficient quality is a major problem. Biotechnological production could be an attractive alternative mode of production. Herein, we explore the potential use of synthetic biology as an alternative strategy for synthesis of cannabinoids 2 in heterologous hosts. We summarize the current knowledge surrounding cannabinoids biosynthesis and present a comprehensive description of the key steps of the genuine and artificial pathway, systems biotechnology needs and platform optimization.
Abstract

D9 -Tetrahydrocannabinol (D9 -THC) attenuates mouse sperm motility and male fecundity

Daniel J Morgan, Charles H Muller, Natalia A Murataeva, Brian J Davis and Ken Mackie
British Journal of Pharmacology (2012) 165 2575–2583
DOI: 10.1111/j.1476-5381.2011.01506.x

Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1 cannabinoid receptor agonist, and D9-tetrahydracannabinol (D9 -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of D9 -THC on male fecundity were determined.
Abstract