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Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.

ABSTRACT: Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpenoids. It has been proposed that cannabinoids act in synergy with terpenoids to produce the entourage effect, a concept used to explain the therapeutic benefits of medicinal cannabis. One molecular explanation for the entourage effect is that the terpenoids augment the actions of cannabinoids at their molecular drug targets in cells. We recently reported that terpenoids commonly found in cannabis do not influence the functional effects of ?⁹-tetrahydrocannabinol (?⁹-THC) on cannabinoid 1 and cannabinoid 2 receptors. The present study aimed to extend on this research by examining whether terpenoids influence the effects of phytocannabinoids and endocannabinoids on human transient receptor potential ankyrin 1 (hTRPA1) and human transient receptor potential vanilloid 1 (hTRPV1) channels heterologously expressed in mammalian cells. Materials and Methods: The activity of terpenoids, phytocannabinoids, and endocannabinoids was assessed in inducible HEK Flp-In T-Rex cells transfected with hTRPA1 and hTRPV1 channels, respectively. Real-time changes in intracellular calcium ([Ca]_i) were measured using the Calcium 5 dye and a FlexStation 3 plate reader. Results: ?-pinene, ?-pinene, ?-caryophyllene, linalool, limonene, ?-myrcene or ?-humulene did not affect [Ca]_i in hTRPA1 and hTRPV1 overexpressing cells. Cinnamaldehyde (CA), ?⁹-THC, and 2-arachidonoylglycerol (2-AG) activated TRPA1 receptors with high efficacy and similar potency (EC₅₀s of ?10??M). Capsaicin and anandamide (AEA) activated TRPV1 receptors with an EC₅₀ of 61?nM and 4.3??M, respectively, but TRPV1 showed no response to ?⁹-THC, cannabidiol, and other minor cannabinoids. Terpenoids did not significantly affect the responses of TRPA1 and TRPV1 receptors to submaximal and maximal concentrations of CA and ?⁹-THC or the endocannabinoids AEA and 2-AG. Discussion: We could not find any evidence that the terpenoids tested here activate TRPA1 and TRPV1 channels or modulate their activation by ?⁹-THC and other agonists, including endocannabinoids.

SUBMITTER: Heblinski M

PROVIDER: S-EPMC7759271 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.

Heblinski Marika M Santiago Marina M Fletcher Charlotte C Stuart Jordyn J Connor Mark M McGregor Iain S IS Arnold Jonathon C JC

Cannabis and cannabinoid research 20201215 4

Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpenoids. It has been proposed that cannabinoids act in synergy with terpenoids to produce the entourage effect, a concept used to explain the therapeutic benefits of medicinal cannabis. One molecular explanation for the entourage effect is that the terpenoids augment the actions of cannabinoids at their molecular drug targets in cells. We recently reported that terpenoids commonly ...[more]

PMID: 33376801

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Project description:Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids may act in concert to elicit therapeutic effects. Cannabinoids such as ?9-tetrahydrocannabinol (?9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2); however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signaling. Therefore, we examined six common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signaling in vitro. Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30?min. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels. Results: ?-Pinene, ?-pinene, ?-caryophyllene, linalool, limonene, and ?-myrcene (up to 30-100??M) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by ?9-THC (10??M): (CB1: control, 59%±7%; with terpenoids (10??M each) 55%±4%; CB2: ?9-THC 16%±5%, with terpenoids (10??M each) 17%±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with ?9-THC and signaling measured continuously over 30?min. Terpenoids did not affect desensitization, after 30?min the control hyperpolarization recovered by 63%±6% in the presence of the terpenoids recovery was 61%±5%. Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signaling of the phytocannabinoid agonist ?9-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signaling pathways that do not involve potassium channels; however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioral effect of Cannabis.

Project description:In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-Δ9-trans-tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings may point to the functional roles of secondary metabolites in Cannabis.

Dataset Information

Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.

Publications

Terpenoids Commonly Found in <i>Cannabis sativa</i> Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.

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