Terpenoids Commonly Found in Cannabis Sativa Do Not Modulate the Actions of Phytocannabinoids Or Endocannabinoids on TRPA1 and TRPV1 Channels
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Cannabis and Cannabinoid Research Volume 5, Number 4, 2020 Mary Ann Liebert, Inc. DOI: 10.1089/can.2019.0099 Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels Marika Heblinski,1,2 Marina Santiago,3 Charlotte Fletcher,1,4 Jordyn Stuart,1,3,4 Mark Connor,3 Iain S. McGregor,1,4 and Jonathon C. Arnold1,2,* Abstract Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpe- noids. 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 en- tourage 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 D9-tetrahydrocannabinol (D9-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 endo- cannabinoids 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: a-pinene, b-pinene, b-caryophyllene, linalool, limonene, b-myrcene or a-humulene did not affect [Ca]i in hTRPA1 and hTRPV1 overexpressing cells. Cinnamaldehyde (CA), D9-THC, and 2-arachidonoylglycerol (2-AG) activated TRPA1 receptors with high efficacy and similar potency (EC50sof*10 lM). Capsaicin and anandamide (AEA) activated 9 TRPV1 receptors with an EC50 of 61 nM and 4.3 lM, respectively, but TRPV1 showed no response to D -THC, canna- bidiol, and other minor cannabinoids. Terpenoids did not significantly affect the responses of TRPA1 and TRPV1 re- ceptors to submaximal and maximal concentrations of CA and D9-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 D9-THC and other agonists, including endocannabinoids. Keywords: terpenoid; TRPA1; TRPV1; phytocannabinoid; endocannabinoid; entourage effect Introduction However, cannabis contains other phytochemicals, in- Cannabis plant matter contains hundreds of diverse cluding various minor phytocannabinoids, as well as bioactive molecules. The major components of canna- flavonoid and terpenoid molecules, which have their bis include phytocannabinoids such as the main psy- own biological activities.1,2 The burgeoning utilization choactive constituent D9-tetrahydrocannabinol (D9- of medicinal cannabis necessitates research that ad- THC) and the nonpsychoactive cannabidiol (CBD).1 dresses the complex polypharmacology of cannabis. 1The Lambert Initiative for Cannabinoid Therapeutics, Brain and Mind Centre, The University of Sydney, Sydney, Australia. 2Faculty of Medicine and Health and School of Medical Sciences, The University of Sydney, Sydney, Australia. 3Faculty of Medicine and Health Sciences, Macquarie University, Macquarie Park, Sydney, Australia. 4Faculty of Science and School of Psychology, The University of Sydney, Sydney, Australia. *Address correspondence to: Jonathon C. Arnold, PhD, The Lambert Initiative for Cannabinoid Therapeutics, Brain and Mind Centre, The University of Sydney, Sydney, NSW 2050, Australia, E-mail: [email protected] ª Marika Heblinski et al. 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 305 306 HEBLINSKI ET AL. Relevant to such research is the ‘‘entourage effect’’ hy- fects of phytocannabinoids at transient receptor poten- pothesis, which posits that the effects of the whole of tial ankyrin 1 (TRPA1) and transient receptor potential the cannabis plant is substantially greater than the vanilloid 1 (TRPV1) channels. sum of its individual components. The ‘‘entourage ef- TRPA1 channels play an important role in pain, fect’’ is often quoted by the growing community of can- itch, allergic cough, and asthma,15 and TRPV1 chan- nabis entrepreneurs and patient advocates to explain the nels are involved in epilepsy, pain, thermoregulation, therapeutic and safety benefits of medicinal cannabis. and itch.15 Phytocannabinoids are known agonists of While few scientific studies have directly addressed these receptors, with D9-THC activating TRPA1, as well the ‘‘entourage effect’’ hypothesis, there is accumulating as CBD and other minor phytocannabinoids being evidence for cannabis plant extracts having effects that reported to activate TRPV1.16 Furthermore, endocanna- cannot simply be attributed to individual cannabis con- binoids affect these receptors, and anandamide (AEA) stituents.3 One early study showed that D9-THC-rich and 2-arachidonoylglycerol (2-AG) are TRPV1 and cannabis extracts had more potent effects than D9- TRPA1 agonists, respectively.17–19 Given that TRPA1 THC administered as a purified substance in humans and TRPV1 receptors appear to have allosteric sites, and animals, implying that other components in the it is possible that entourage effects are mediated by ter- extract might enhance the effects of D9-THC.4 Consis- penoid allosteric modulation of cannabinoid actions tent with this finding more recent studies showed that at these receptors.20–22 In the present study we aimed D9-THC-rich cannabis extracts had greater anticancer to assess whether seven terpenoids most commonly effects than purified D9-THC on human cancer cells found in cannabis have activity at TRPV1 and TRPA1 in vitro and in vivo.5,6 channels alone, as well as observing whether they mod- One means through which ‘‘entourage effects’’ may ulate the effects of phytocannabinoids and endocanna- occur is via the interaction of individual components binoids on these receptors. of the cannabis plant. Interplay between different can- nabinoids provides one possibility, and numerous Materials and Methods studies have examined interactions between the phyto- Cell culture cannabinoids CBD and D9-THC. These studies have For this study, we used HEK293 cells as an expression sys- revealed great complexity in the interaction, with tem as they have been used extensively in previous studies CBD synergistically enhancing or inhibiting the effects assessing TRPV1 and TRPA1 channel signaling.23–25 Flp- of D9-THC dependent on the measured variable and In T-Rex HEK 293 cells (Life Technologies, Mulgrave, the frequency of dosing.7–10 It has also been hypothes- Victoria, Australia) were stably transfected with human ised that the varying effects of different cannabis che- TRPA1 (hTRPA1) or human TRPV1 (hTRPV1) cDNA movars might be due to variation in their terpene (GenScript, Piscataway, NJ) and cultivated in Dulbecco’s content, which can reach concentrations of 3.5% and modified Eagle’s medium supplemented with 10% fetal 10% in flower and trichomes, respectively.11–13 How- bovine serum (FBS), 100 U mLÀ1 penicillin, and 100 lg ever, few studies have addressed this hypothesis; thus, mLÀ1 streptomycin. Medium for transfected cells was the present study sought to advance the evidence supplemented with hygromycin B 80 lgmLÀ1 and blasti- base on potential terpenoid–cannabinoid interactions. cidin S 10 lgmLÀ1. Medium for Flp-In T-Rex HEK293 One means to deconvolute the complex actions of cells was supplemented with zeocin 100 lgmLÀ1 and whole plant cannabis formulations on human biology blasticidin S 15 lgmLÀ1. is to assess whether phytochemical synergy occurs at Cells were incubated in 5% CO2 at 37°C in a humid- the level of drug targets heterologously expressed in ified atmosphere. Cells were grown in flasks with a sur- mammalian cells. We recently used this approach by face area of 75 mm2, once at optimum confluence examining whether several terpenoids influenced the (*90%); cells were trypsinized and seeded into a 9 agonist activity of D -THC at cannabinoid 1 (CB1) poly-D-lysine coated black, clear-bottomed 96-well 14 and 2 (CB2) receptors expressed in AtT20 cells. The plate (Corning, Castle Hill, NSW, Australia) at a den- terpenoids analyzed failed to affect the actions of D9- sity of 110,000–130,000 cells per well in L15 medium THC on these receptors; however, this doesn’t preclude supplemented with 1% FBS, 15 mM glucose, 100 U interactions at other cannabinoid drug targets. In the penicillin, and 100 lgmLÀ1 streptomycin. The cells present study we aimed to extend on our prior research were plated in a volume of 80 lL and were incubated by examining whether the terpenoids modulate the ef- overnight in a humidified chamber at 37°C and EFFECTS OF TERPENOIDS ON TRPA1 AND TRPV1 307 ambient CO2. Expression of the hTRPA1 and hTRPV1 and 96% (a -humulene). All tissue culture reagents were channel was induced 4.5 h before experimentation by from Sigma-Aldrich, Life Technologies (Mulgrave, Vic- addition of 2 lgmLÀ1 tetracycline per well. toria, Australia) or InvivoGen (San Diego,