Cannabidiol selectively inhibits the contraction of rat small resistance arteries: possible role for CGRP and voltage-gated calcium channels

Amna C Mazeh, James A Angus, Christine E Wright1

Abstract

The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis sativa, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 µM) or vehicle and concentrationcontraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, α-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin gene-related peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPARγ or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 µM significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 µM) was largely without effect in bronchi, atria and hemidiaphragm, but 100 µM attenuated maximum contractions in vasa deferentia. Cannabidiol’s effects in the clinical range (1-3 µM) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

Keywords: Cannabidiol; calcitonin gene-related peptide; L-type voltage-gated calcium channels; vascular smooth muscle contraction; small mesenteric arteries.

1. Introduction

Cannabidiol, the non-psychoactive major component of Cannabis sativa, is increasingly prescribed for a range of medical applications, including as an anxiolytic, anti-inflammatory, antiemetic, analgesic, antipsychotic and anticonvulsant (Mechoulam et al., 2002). Additionally, in 2018 the USA Food and Drug Administration approved an oral solution of cannabidiol (Epidiolex®, GW Pharmaceuticals) for the treatment of seizures associated with rare forms of childhood epilepsy following a clinical trial in children with Dravet or LennoxGastaut syndrome (Devinsky et al., 2017; Devinsky et al., 2018a). In a dose-ranging safety trial of cannabidiol 5, 10 and 20 mg/kg/day, plasma levels of cannabidiol at day 22 were approximately 110, 220 and 380 ng/ml which equate to concentrations of 0.35, 0.70 and 1.2 µM, respectively (Devinsky et al., 2018b). A similar cannabidiol plasma concentration of 1.4 µM was measured in children with epilepsy after 8 weeks of cannabidiol 25 mg/kg/day (Geffrey et al., 2015). No cardiovascular effects were reported in either study (Devinsky et al., 2018b; Geffrey et al., 2015). In healthy adult volunteers (average body weight 77 kg), cannabidiol 600 mg (7.8 mg/kg) caused a fall in mean arterial pressure (≈5 mmHg) and increased heart rate (+10 beats/min) 2 h after oral dosing (Jadoon et al., 2017).
In studies with isolated arteries such as rat aorta, cannabidiol 10 µM enhanced vasorelaxation to acetylcholine in arteries preincubated with cannabidiol for 2 h (Stanley et al., 2013) and induced time-dependent vasorelaxation of precontracted arteries acutely treated with cannabidiol for 2 h (O’Sullivan et al., 2009). Further, in rat isolated small mesenteric arteries precontracted with phenylephrine, acute cannabidiol administration caused maximum relaxation with a pIC50 of 5.66 ± 0.06 (Baranowska-Kuczko et al., 2020; Offertaler et al., 2003). This full relaxation of small mesenteric resistance arteries has also been reported for anandamide, while the larger superior mesenteric artery only relaxed by 40% (Randall et al., 2004). In human isolated large pulmonary arteries precontracted with U46619, acute cannabidiol 10 µM did not cause relaxation. Further, in the same paper, in rat small mesenteric arteries (250 µm i.d.) pretreatment with cannabidiol 1 µM had no effect on phenylephrine concentration-response curves (Baranowska-Kuczko et al., 2020). The general pharmacological properties of cannabidiol have recently been reviewed (McPartland et al., 2015); its Appendix S2 is a detailed literature review regarding the interaction of cannabidiol with many pharmacological targets.
In the present work, we aimed to quantify the effects of clinically used concentrations of cannabidiol on a wide range of contractile agents in rat small resistance arteries and compared these with larger arteries. We have also explored the mechanism of action of cannabidiol in small resistance arteries. Finally, we compared the vascular action of cannabidiol with the contraction of various non-vascular tissues – bronchial, urogenital, cardiac and skeletal muscles.
Our results show that cannabidiol 1-3 µM (i.e. in the clinical range) significantly inhibited the contraction of small resistance arteries through a combination of mechanisms that include calcitonin gene-related peptide (CGRP) and L-type voltage-operated calcium channels. Interestingly, these effects appear to be specific to small resistance arteries as no large artery or non-vascular tissue appeared to be sensitive to cannabidiol. These findings have implications for targeting the therapeutic effects of cannabidiol.

2. Materials and methods

The Animal Ethics Committee of the University of Melbourne approved experiments (approval #10246) in accordance with The Australian Code for the care and use of animals for scientific purposes (8th edition, 2013, National Health and Medical Research Council, Canberra). Male Sprague-Dawley rats (10-12 weeks old, Biomedical Animal Facility, Melbourne, Australia) were used in this study and housed in groups of 3-4 in standard cages under constant climatic conditions (21ºC, 12 h light/dark cycle), with food and water ad libitum. Rats (250-350 g) were deeply anaesthetised by inhalation of 5% isoflurane in 100% oxygen and killed by a rapid cut through the spinal cord. All experiments were performed in KrebsHenseleit physiological salt solution (PSS) with the following composition (mM): NaCl 119, KCl 4.7, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, EDTA 0.026, MgSO4 1.2 and glucose 5.5 for bronchi, mesenteric, saphenous and tail arteries and 11 for vasa deferentia, atria and hemidiaphragm. The PSS was oxygenated with carbogen (95% O2 and 5% CO2) at pH 7.4.

2.1 Isolated arteries

For isolation of mesenteric arteries, the abdomen was opened, the jejunum removed, and its attached vascular fan was pinned out on a Silastic-bottomed petri dish filled with oxygenated PSS. The superior mesenteric artery (i.d. 888 ± 95 µm) and third- or fourthorder mesenteric artery branches (i.d. 200-350 µm) were trimmed from attached tissue. For isolation of saphenous arteries, the muscle over the right hindlimb was exposed and fresh PSS was added while dissecting out large and small saphenous arteries. The saphenous arteries were isolated by dissecting out the left first-order large saphenous artery (i.d. 623 ± 18 µm) and the right second-order small saphenous artery (i.d. 288 ± 12 µm). For isolation of tail arteries, the tail was pinned dorsal side up on a Silastic-covered petri dish filled with PSS, the skin opened to expose the midventral artery (i.d. 923 ± 30 µm) which was carefully lifted and dissected out.
Small segments (2 mm long) of each artery type were mounted on 40 µm diameter stainless-steel wires connected to force transducers in myograph chambers (Model 610M and 620M; Danish Myo Technology, Aarhus Denmark). Responses were captured by a Powerlab (4/30 or 8/35) A/D converter (ADInstruments, Bella Vista, NSW, Australia) and measured on a Mac computer running Labchart 7 Pro data acquisition software (ADInstruments). After an equilibration period in PSS at 37°C, arteries were normalised by stepwise stretching, allowing for the calculation of the artery i.d. under an equivalent transmural pressure of 100 mmHg (D100). The artery diameter was then partially decreased to 0.9D100, i.e. a transmural pressure equivalent to about 60 mmHg to allow for maximum contraction to develop when a contractile agent is applied (described in more detail by
Angus and Wright, 2000). This passive force remained for the rest of the experiment. After a 30 min equilibration, artery viability was tested. Arteries were exposed to a potassium-rich depolarising solution (KCl replacing NaCl, termed KPSS) and noradrenaline 10 µM for 2 min before being washed with PSS. After 10 min (or until the contractile force was back to baseline), arteries were re-exposed to KPSS for 2 min to obtain the maximal contraction and then washed with PSS. All responses were expressed as a percentage of the KPSS response in each artery. After 10 min equilibration, the arteries were precontracted with noradrenaline to ≈80% KPSS contraction, followed by the addition of acetylcholine 1 µM to test the integrity of the endothelium (only arteries that relaxed by >70% were used in experiments). The arteries were then washed with PSS and exposed to one of the following protocols.

2.1.1 Isolated artery protocols

2.1.1.1 Concentration-response curves to different contractile agents

Cannabidiol (0.3, 1 and 3 µM; each dissolved in DMSO 0.1%) or vehicle (DMSO 0.1%) were added to the baths and equilibrated for 30 min, followed by cumulative additions of half-log increments of a contractile agent. We chose this cannabidiol pretreatment protocol rather than applying cannabidiol to a precontracted artery to allow 30 min incubation of a single concentration of cannabidiol. We also found that the lack of stability of precontracted small mesenteric arteries made this alternate latter protocol difficult to use. Large saphenous, mesenteric and tail arteries were exposed to the contractile agonist methoxamine (0.01100 µM), while small saphenous arteries were exposed to either methoxamine (0.01-300 µM) or 5-HT (0.01-10 µM). In small mesenteric arteries cumulative concentrationcontraction responses to endothelin-1 (0.1-100 nM), arginine vasopressin (0.01-30 nM), U46619 (0.001-3 µM), methoxamine (0.01-100 µM), 5-HT (0.01-10 µM) or α-methyl-5-HT (0.01-10 µM) were constructed. Only a single concentration-response curve to an agent was constructed in each artery preparation. Separate arteries were incubated with CGRP (small mesenteric arteries 0.1-100 nM or superior mesenteric arteries 100 nM) or vehicle (MilliQ water) for 30 min, followed by cumulative additions of methoxamine (0.01-100 µM).

2.1.1.2 Potential mechanisms of action of cannabidiol

To explore the mechanism of action of cannabidiol, small mesenteric arteries were equilibrated with one of the following treatments for 30 min (except B) (literature supporting the chosen treatment concentrations shown): (A) olcegepant 1 µM (CGRP receptor antagonist) (Sheykhzade et al., 2017); (B) capsaicin 1 µM (transient receptor potential cation channel 1 (TRPV1) receptor desensitiser; arteries incubated for 1 h followed by a wash out of capsaicin) (Voets et al., 2004); (C) L-NAME 100 µM (nitric oxide synthase (NOS) inhibitor) (Angus et al., 2017); (D) indomethacin 3 µM (cyclooxygenase inhibitor) (Kassab et al., 2017); (E) combination of olcegepant 1 µM, L-NAME 100 µM and indomethacin 3 µM; (F) GW9662 1 µM (peroxisome proliferator-activated receptor γ (PPARγ) antagonist) (Leesnitzer et al., 2002); (G) SR141716 300 nM (cannabinoid receptor type 1 (CB1) antagonist) (Christopoulos et al., 2001); (H) SR144528 30 nM (CB2 receptor antagonist) (Abood et al., 2019); or (I) O-1918 3 µM (inhibitor of endothelium-mediated vasorelaxation of cannabinoids) (McHugh et al., 2010). After treatment incubation, arteries were incubated with cannabidiol (1 or 3 µM) or vehicle (DMSO 0.1%) for 30 min and then a concentrationcontraction curve was constructed with cumulative additions of either methoxamine or 5-HT (0.01-100 µM). Small saphenous arteries were equilibrated with olcegepant (1 µM) followed by the incubation of cannabidiol (3 µM) or vehicle (DMSO 0.1%) for 30 min and cumulative additions of 5-HT (0.01-30 µM). We chose to examine the mechanism of action of cannabidiol in detail against methoxamine as it is a selective α1-adrenoceptor agonist that mimics sympathetic vasomotor constriction and 5-HT as a different vasoactive amine acting at mainly 5-HT1A receptors as a vasoconstrictor of small arteries.

2.1.1.3 Potential role of calcium

To study the role of extracellular calcium, mesenteric arteries were washed with calciumfree PSS and allowed to equilibrate for 10 min. The intracellular calcium storage was depleted by repeatedly exposing the arteries to KCl (80 mM) or methoxamine (10 µM) for 5 min before washing the arteries with calcium-free PSS. This was followed by the incubation of arteries with cannabidiol (0.3, 1 and 3 µM), CGRP (100 nM) or vehicle (DMSO 0.1%) for 30 min. Arteries treated with cannabidiol or vehicle were thereafter incubated with either KCl (80 mM) or methoxamine (10 µM) for 5 min to induce the opening of calcium channels, and CGRP-treated arteries were incubated with methoxamine (10 µM) for 5 min, prior to construction of a CaCl2 concentration-contraction curve (cumulative additions, 0.01-10 mM). To assess the involvement of L-type voltage-operated calcium channels, arteries were incubated in normal PSS with cannabidiol (0.3, 1 and 3 µM) or vehicle (DMSO 0.1%) for 30 min prior to the addition of KCl 20 mM and cumulative additions of the L-type voltageoperated calcium channel agonist Bay K8644 (0.1-300 nM).

2.2 Isolated non-vascular smooth muscle: bronchi and vasa deferentia

2.2.1 Bronchi

The left lung lobe was isolated and pinned on a Silastic-bottomed petri dish with oxygenated PSS, dorsal side up. The main bronchus was cut, exposing the opening of secondary bronchi; tissue surrounding the bronchi was removed and the ends of bronchi were lifted and separated from attached pulmonary arteries. Small segments of bronchi (2 mm in length) were mounted in myographs, as described above. After an equilibration period in PSS at 37°C, the bronchi were stretched to 1.5 mN, followed by a re-stretch 10 min later. After a 10 min equilibration of stretched bronchi, tissue viability was tested. The bronchi were exposed to KPSS and carbachol (100 µM) for 2 min, before being washed with PSS. All responses were expressed as % KPSS + carbachol (100 µM) maximum contraction in each tissue. Following the viability test, cannabidiol (10, 30 and 100 µM) or vehicle (DMSO 1%) was added to the baths and equilibrated for 1 h, followed by cumulative additions of halflog increments of carbachol (0.01-300 µM).

2.2.2 Vasa deferentia

The whole vasa deferentia were excised by pushing the testis up towards the abdomen and then cutting it loose from the epididymis towards the prostate end. It was pinned down to a Silastic-bottomed petri dish with oxygenated PSS, tied at either side with a silk thread and trimmed to an approximate length of 2 cm. The prostatic end was tied to a stainless-steel hook on a fixed acrylic organ bath leg, while the epididymal end was tied uppermost to a stainless-steel hook attached to a Grass FT03C isometric force transducer (Grass Instruments, Quincy, MA, USA) connected to a Powerlab 8/35 data acquisition system (ADInstruments). The organ bath leg was adjusted vertically with an attached micrometer (Mitutoyo Manufacturing Co., Kawasaki, Japan). The responses were measured on a computer running Labchart 7 Pro data acquisition software (ADInstruments). Tissues were suspended in 5 ml organ baths containing PSS oxygenated with 95% O2 and 5% CO2 at 37°C, stretched to 2 g force, washed and equilibrated for 30 min. The viability of the tissues was tested by exposing the tissues to noradrenaline (10 µM) for 2 min and then washed. After 10 min of equilibration the tissues were incubated with either cannabidiol (30 and 100 µM) or vehicle (DMSO 1%) for 1 h, followed by cumulative additions of half-log increments of noradrenaline (0.01-300 µM).

2.3 Isolated cardiac muscle: left and right atria

The heart was isolated and placed in a Silastic-bottomed petri dish filled with oxygenated PSS. The atria were isolated, connective tissue removed and left and right atria separated. Metal hooks were placed on each side of the atrium. One side of the tissue was pierced with a stainless-steel hook attached to a Grass FT03C isometric force transducer connected to a bridge amplifier and a Powerlab 8/35data acquisition system (ADInstruments) while the other side was hooked to a fixed acrylic organ bath leg between two parallel platinum field electrodes. Atria were suspended in 25 ml organ baths containing PSS oxygenated with 95% O2 and 5% CO2 at 37°C. The tissues were stretched to 1 g force, followed by a restretch to 1 g after 10 min, followed by a wash. The left atrium rested between two punctate platinum electrodes to enable the delivery of a train of field pulses (0.5 Hz, 0.2 ms duration) by a Grass S88 stimulator via a Grass stimulus isolation unit (SIU5). This stimulus frequency was chosen to conserve cardiac tissue viability as it is a >1 mm thick muscle, requiring oxygenation by diffusion from the bathing solution. The voltage of the stimulation was gradually increased until the threshold voltage was reached, followed by 150% increase of the threshold voltage. Both atria were equilibrated for 30 min before their viability was tested by exposing the tissues to isoprenaline (1 and 0.1 µM, left and right atria, respectively) for 2 min and then washed. All responses were expressed as % of the maximum isoprenaline response. Once responses returned to baseline, atria were incubated with either cannabidiol (10-100 µM) or vehicle (DMSO 1%) for 1 h, followed by cumulative additions of half-log increments of isoprenaline (0.01-1000 nM).

2.4 Isolated skeletal muscle: phrenic nerve-hemidiaphragm

The upper ribcage was removed caudally to the thymus exposing the phrenic nerves and their attachments to the diaphragm. The thymus ends of the nerves were tied with a silk thread and then cut free from attached connective tissues, ensuring remaining attachments to the diaphragm. The diaphragm with attached phrenic nerves was isolated from connective tissue and transferred to a Silastic-bottomed petri dish filled with oxygenated PSS. The diaphragm was bisected along the midline, creating two separate hemidiaphragms with a centrally attached phrenic nerve. The proximal end of the tissue was tied to a stainless-steel hook attached to a Grass FT03C isometric force transducer connected to a Powerlab 8/35 data acquisition system, while the distal end was tied to a stainless-steel hook on a fixed acrylic organ bath leg. The phrenic nerve was threaded through a platinum electrode. The responses were measured on a computer running Labchart 7 Pro data acquisition software. Tissues were suspended in 15 ml organ baths containing PSS oxygenated with 95% O2 and 5% CO2 at 32°C. The tissues were stretched to 2 g force, followed by a re-stretch to 2 g after 10 min and equilibrated for 20 min. Trains of field pulses (4 V, 0.1 Hz, 0.2 ms duration) were delivered by a Grass S88 stimulator via a Grass stimulus isolation unit (SIU5) and increased to 150% of the voltage threshold (100%) of the individual tissue for 30 min before the tissues were incubated with a single addition of either cannabidiol 30 µM or DMSO (0.3%) for 2 h.

2.5 Data and statistical analyses

All data are expressed as the mean ± S.E.M. of n experiments (each tissue from a separate rat). Contractile responses in arteries are expressed as the percentage of the maximum reference contraction to KPSS (%KPSS) within each artery. The individual data points showing the correlation between artery internal diameter and the inhibition of contraction induced by cannabidiol (3 µM) are expressed as the % decrease in contraction compared to maximum contraction to KPSS, in each artery. Responses in bronchi and left atria are expressed as the % of the maximum response to KPSS + carbachol (100 µM) or isoprenaline (1 µM), respectively, within each tissue. Responses in right atria are given as the % of the maximum tachycardia to isoprenaline (0.1 µM). Contractions of vasa deferentia or hemidiaphragms are given as the absolute increase in force from baseline tone.
Data were plotted and analysed using Prism 8 (GraphPad Software, La Jolla, CA, USA). Individual sigmoidal agonist concentration-response curves were fitted and the pEC50 (–log of the concentration (M) of agonist required to elicit the half-maximal response, EC50) and Rmax (maximum response) determined for each tissue. For comparison of pEC50 or Rmax values between >2 treatment groups, repeated measures one-way ANOVA with Dunnett’s post hoc test for multiple pairwise comparisons was performed. Two groups were compared using a Student’s unpaired t test. Statistical significance was taken as P < 0.05.

2.6 Drugs

Drugs used were: Acetylcholine bromide; 8-methyl-N-vanillyl-6-nonenamide (capsaicin); carbamoylcholine chloride (carbachol); GW9662 (2-chloro-5-nitro-N-phenylbenzamide); indomethacin; 5-hydroxytryptamine (5-HT) creatinine sulfate; (-)-isoproterenol (+)-bitartrate (isoprenaline); L-nitro arginine methyl ester (L-NAME); methoxamine hydrochloride; αmethyl-5-hydroxytryptamine maleate; (-)-noradrenaline bitartrate (all from Sigma-Aldrich, St Louis, MO, USA); (-)-cannabidiol; O-1918; rimonabant (SR141716); SR144528; U46619 (all from Cayman Chemical, Ann Arbor, MI, USA); Arg8-vasopressin (AVP; Aviva Systems Biology, San Diego, CA, USA); calcitonin gene-related peptide (CGRP); endothelin-1 (Auspep, Melbourne, VIC, Australia); Bay K8644 (Bayer, Leverkusen, Germany); and BIBN 4096 (olcegepant; Tocris Bioscience, Bristol, UK). Cannabidiol, capsaicin, GW9662, O-1918, olcegepant, SR141716, SR144528 and U46619 were dissolved in DMSO. The other drugs were dissolved in MilliQ water apart from indomethacin that was dissolved in 0.1 M Na2CO3 in addition to water. All aliquots were stored at -20°C as single-use 10-4-10-1 M aliquots.

3. Results

3.1 Effects of cannabidiol on the contraction of small mesenteric arteries

Cannabidiol 3 µM inhibited the contraction of rat isolated small mesenteric arteries to each of the contractile agents tested (endothelin-1, arginine vasopressin, U46619, methoxamine, 5-HT and α-methyl-5-HT) with marked attenuation of the maximum response (Rmax) to each agent (Fig. 1 and Table 1). With endothelin-1, cannabidiol 3 µM caused a small 2.6-fold rightward shift in the agonist pEC50 (P < 0.05; Fig. 1A) with a 55% decrease in Rmax (P < 0.0001); lower cannabidiol concentrations of 0.3-1 µM had no effect. Qualitatively similar findings were observed with arginine vasopressin and U46619 (Figs. 1B-C) where the agonist contraction curves were near flattened (Rmax -69% and -82% compared with the vehicle group, respectively; P < 0.0001, Table 1) by incubation with cannabidiol 3 µM; pEC50 values could not be determined in most tissues. Concentration-contraction response curves to 5-HT (Fig. 1E) were already completely abolished by cannabidiol at 1 µM. In methoxamine- or α-methyl-5-HT-contracted arteries, cannabidiol 0.3, 1 and 3 µM caused a concentration-dependent rightward shift and collapse of the response curves (Figs. 1D and F). Cannabidiol 0.3 µM pretreatment resulted in small 3.7-fold and 2.2-fold increases in methoxamine and α-methyl-5-HT pEC50 values, respectively (P < 0.05, Table 1), while cannabidiol 1 µM shifted methoxamine curves by 9.1-fold, all with no significant decrease in Rmax; contraction curves to both agents were virtually abolished with cannabidiol 3 µM, similar to the other tested agents, except for endothelin-1 (Fig. 1 and Table 1).

3.2 Potential mechanisms of action of cannabidiol in small mesenteric arteries

To investigate the potential mechanisms of action of cannabidiol, arteries were coincubated with receptor and/or enzyme antagonists prior to incubation with vehicle or cannabidiol followed by the construction of concentration-contraction curves to methoxamine (Fig. 2) or 5-HT (Fig. 3). In the presence of any of the antagonists tested, the methoxamine or 5-HT pEC50 and Rmax values of vehicle-treated arteries were comparable to the Control (no co-incubation with antagonist) vehicle group (Table 2; P > 0.1, one-way ANOVA with Dunnett’s post hoc test).

3.2.1 Involvement of CGRP and endothelial factors

The CGRP inhibitor olcegepant (1 µM) partly reversed the effects of cannabidiol on methoxamine- and 5-HT-contracted arteries (Figs. 2A and 3A; Table 2). With olcegepant coincubation, the methoxamine concentration-contraction curve in the presence of cannabidiol 3 µM was shifted 3.7-fold to the right of the vehicle control curve, which was like the effects observed with cannabidiol alone (Fig. 2A and Table 2). However, the methoxamine Rmax was greater with olcegepant and cannabidiol co-incubation (52% of the respective vehicle control group; P = 0.004) compared with cannabidiol alone (19% of control). In 5-HT-contracted arteries, olcegepant substantially recovered responses with a 3.5-fold rightward shift in the presence of cannabidiol 1 µM (P = 0.0098) and Rmax values tending to be decreased but not significantly different from the vehicle control group (P = 0.072; Fig. 3A and Table 2).
The effects of cannabidiol 3 µM on the contractions to methoxamine persisted in the presence of the capsaicin (1 µM; TRPV1 desensitiser), L-NAME (100 µM; NOS inhibitor) and indomethacin (3 µM; cyclooxygenase inhibitor) (Fig. 2B-D and Table 2). In contrast, contractions to 5-HT in the presence of cannabidiol 1 µM were partly reversed (in a similar manner to the effects of olcegepant) when coincubated with capsaicin, L-NAME or indomethacin (Fig. 3B-D). The coincubation of olcegepant, L-NAME and indomethacin in combination recovered the agonist contraction curves to a greater extent than either antagonist alone for the methoxamine or 5-HT contraction curves with significant increases in Rmax compared with cannabidiol alone (Figs. 2E and 3E; P < 0.05, Table 2).
To further elucidate a potential role for endogenous CGRP in the effects of cannabidiol we studied the interaction of exogenous CGRP with methoxamine (Fig. 4). CGRP 1-100 nM caused concentration-dependent rightward shifts of 4.3-19.9-fold of methoxamine concentration-contraction response curves (P < 0.05) and decrease in the Rmax (to 52% of the vehicle group with CGRP 100 nM; P = 0.0005, Fig. 4).

3.2.2 Involvement of PPARγ and cannabinoid receptors

The cannabidiol-induced inhibition of contraction to methoxamine or 5-HT was unaffected by the presence of the PPARγ antagonist GW9662 1 µM (Figs. 2F and 3F; Table 2) or the CB1 receptor antagonist SR141716 (300 nM) (Figs. 2G and 3G). The CB2 receptor antagonist SR144528 (30 nM) was without significant effect on the inhibition of methoxamine contractions caused by cannabidiol (3 µM) (Fig. 2H), however, SR144528 pretreatment did partially restore contractions to 5-HT in the presence of cannabidiol 1 µM (Fig. 3H and Table 2). O-1918 (3 µM), an inhibitor of endothelium-dependent vasorelaxation to cannabinoids, had no effect on the cannabidiol-induced inhibition of contraction to methoxamine (Fig. 2I), but interestingly, it restored the maximum contraction (Rmax) to 5-HT in the presence of cannabidiol to a level not different from the vehicle control (Fig. 3I and Table 2), with a 4.8-fold rightward shift of the 5-HT contraction-response curve.

3.2.3 Involvement of methoxamine- and potassium-induced calcium entry

Cannabidiol 1 and 3 µM inhibited calcium-dependent contractions in small mesenteric arteries (Fig. 5). Cannabidiol caused a concentration-dependent inhibition of contraction to CaCl2 in both KCl (80 mM) or methoxamine (10 µM) pretreated arteries. In KCl pretreated arteries, cannabidiol 1 µM caused a 2.6-fold dextral shift of the CaCl2 concentrationcontraction response curve with a decrease in Rmax of 44% compared with the vehicle group (CaCl2 pEC50, vehicle 3.19 ± 0.09 and cannabidiol 1 µM 2.77 ± 0.05, P = 0.0007; Rmax, vehicle 71 ± 6% and cannabidiol 1 µM 40 ± 8%, P = 0.0011; Fig. 5A). In methoxamine 10 µM pretreated arteries, cannabidiol 1 µM caused a small 1.5-fold rightward shift in the CaCl2 curve without a significant decrease in Rmax (CaCl2 pEC50, vehicle 3.79 ± 0.04 and cannabidiol 1 µM 3.60 ± 0.04, P = 0.03; Fig. 5B). In KCl- or methoxamine-pretreated arteries, the higher concentration of cannabidiol (3 µM) caused a near complete inhibition of contraction to CaCl2 (Fig. 5A-B). Cannabidiol 1-3 µM concentration-dependently attenuated the maximum contractions to Bay K8644, an L-type voltage-operated calcium channel agonist (cannabidiol 1 µM -59% (P = 0.0004) and cannabidiol 3 µM -90% (P < 0.0001) compared with vehicle group Rmax; Fig. 5C). The Bay K8644 pEC50 tended to be 2.5-fold right-shifted in the presence of cannabidiol 1 µM, but this did not reach significance (P = 0.28); pEC50 values could not be determined with cannabidiol 3 µM (Fig. 5C).
Similarly to cannabidiol, CGRP (100 nM) inhibited contractions to CaCl2 in methoxamine (10 µM) pretreated arteries, causing a 2.1-fold dextral shift of contraction responses to CaCl2, while also attenuating the Rmax (CaCl2 pEC50, vehicle 3.95 ± 0.07 and CGRP 100 nM 3.62 ± 0.09, P = 0.03; Rmax in CGRP group -69% of vehicle group, P = 0.0004; Fig. 5D).

3.3 A comparison of the effects of cannabidiol in small and large arteries

3.3.1 Cannabidiol effects on methoxamine and 5-HT responses in small and large arteries

The contractions to methoxamine or 5-HT of small saphenous arteries were inhibited by cannabidiol 3 µM (Fig. 6A-B) in the same manner as observed in small mesenteric arteries (Fig. 1). In small saphenous arteries cannabidiol 3 µM caused a 5.3-fold rightward shift and attenuation of Rmax (-66% of vehicle Rmax, P < 0.0001) in methoxamine concentrationcontraction curves (methoxamine pEC50, vehicle 5.11 ± 0.15 and cannabidiol 3 µM 4.39 ± 0.01, P = 0.004; Fig. 6A). Compared to the small mesenteric arteries (Fig. 1D), the saphenous arteries were less sensitive to cannabidiol <3 µM, while also being relatively insensitive to methoxamine. Methoxamine was 10.5 times more potent in small mesenteric arteries compared to the saphenous arteries. In small saphenous arteries, like in small mesenteric arteries, cannabidiol inhibited contractions to 5-HT to a greater degree than to methoxamine (Fig. 6B). Cannabidiol 3 µM caused a decrease in Rmax of -87% of that in the vehicle group (P < 0.0001). In contrast to methoxamine, 5-HT showed a similar potency in small mesenteric and small saphenous arteries.
In large saphenous arteries, only cannabidiol 3 µM had a significant effect on methoxamine contraction curves with a 5.2-fold rightward shift and small 10% decrease in Rmax (Fig. 6C; methoxamine pEC50, vehicle 6.23 ± 0.03 and cannabidiol 3 µM 5.51 ± 0.09, P < 0.0001; Rmax, vehicle 102 ± 2% and cannabidiol 3 µM 91 ± 3%, P = 0.039). In large superior mesenteric and tail arteries, cannabidiol 0.3-3 µM was without effect on methoxamine contraction responses (Fig. 6D-E).
In general, there was a negative correlation between artery internal diameter and the maximum inhibition of contraction to methoxamine by cannabidiol 3 µM (Fig. 7; r2 = 0.7), where the smaller resistance arteries were more sensitive to cannabidiol. In addition, methoxamine was 2 times more potent in small mesenteric arteries compared to the superior mesenteric arteries.

3.3.2 Involvement of CGRP in the effects of cannabidiol in small saphenous and superior mesenteric arteries

To investigate the role of CGRP in the effects of cannabidiol in small saphenous arteries, tissues were incubated with the CGRP antagonist olcegepant (1 µM; Fig. 8A). Olcegepant partly reversed the inhibitory effects of cannabidiol 3 µM, allowing 4 out of 6 arteries to concentration-dependently contract to 5-HT (pEC50, vehicle 6.31 ± 0.20 and cannabidiol 6.06 ± 0.08, P = 0.37), with an attenuation in Rmax of -45% compared to the vehicle group (P = 0.03), in marked contrast to the almost complete lack of response to 5-HT when vessels were treated with cannabidiol 3 µM alone. Olcegepant pretreatment did not change the sensitivity or Rmax of 5-HT contractions in the presence of vehicle (Figs. 8A and 6B; pEC50 P = 0.74 and Rmax P = 0.65). Like cannabidiol 3 µM (Fig. 6D), CGRP 100 nM had no effect on methoxamine concentration-contraction response curves in superior mesenteric arteries (methoxamine pEC50, vehicle 5.52 ± 0.13 and CGRP 5.20 ± 0.09, P = 0.09; Rmax, vehicle 102 ± 5% and CGRP 86 ± 6%, P = 0.06; Fig. 8B).

3.4 Effects of cannabidiol on non-vascular muscle

To test whether the effects of cannabidiol are limited to vascular smooth muscle, we investigated the effects of cannabidiol (10-100 µM) on various non-vascular tissues, including bronchial, urogenital, cardiac and skeletal muscle (Fig. 9). The contractions of bronchial smooth muscle and vasa deferentia to carbachol and noradrenaline, respectively, were unaffected by lower concentrations of cannabidiol (10 and/or 30 µM; Fig. 9A-B). Cannabidiol 100 µM decreased the maximum contraction of bronchi by 20% and vasa deferentia by 45% (P < 0.05), without shifting the agonist concentration-contraction curves.
In contrast to the non-vascular smooth muscle tissues, the contraction of left atria to isoprenaline was sensitive to the lower concentrations of cannabidiol 10 and 30 µM but resistant to cannabidiol 100 µM (isoprenaline pEC50, vehicle 8.60 ± 0.07, cannabidiol 10 µM 7.94 ± 0.12 (P < 0.0001), cannabidiol 30 µM 8.24 ± 0.05 (P = 0.004) and cannabidiol 100 µM 8.45 ± 0.05 (P = 0.35); Fig. 9C); Rmax was unaffected by cannabidiol. Isoprenalineinduced increases in right atrial rate were not affected by cannabidiol 10-100 µM (Fig. 9D; pEC50 P > 0.11). In ≈30% of experiments, incubation of cannabidiol 100 µM completely stopped the passive beating of the right atria before the cumulative additions of isoprenaline. The skeletal muscle preparation of the hemidiaphragm demonstrated similar insensitivity to cannabidiol as the other non-vascular muscle tissues. Cannabidiol 30 µM had no effect on the electrically induced contractions of the hemidiaphragm (Fig. 9E).

4. Discussion

Our study has quantified the potent effect of (-)-cannabidiol, at therapeutic concentrations (1-3 µM), in inhibiting the contraction by multiple agents of small resistance arteries, but not large conduit arteries. We also show that non-vascular smooth muscle is generally unaffected by cannabidiol even at 10-100 times the therapeutic plasma level. The sensitivity of the small resistance arteries to cannabidiol involves CGRP and voltageoperated calcium channels.

4.1 Small resistance arteries

In the rat small mesenteric artery mounted and stretched to a passive tension that reflects its diameter at a transmural pressure of about 60 mmHg, there is generally no active tension present. Thus, any pretreatment, such as cannabidiol, or other agents we used here that may induce relaxation cannot be observed on this passive tension. However, when the artery is contracted with the concentration-response curve to methoxamine or 5-HT the interaction with the pretreatment becomes obvious as a right-shift of the contraction curve or depression of the Rmax depending upon the interactions between the constrictor stimulus and the opposing relaxation stimulus from the pretreatment. In small arteries, preincubated with cannabidiol, the sensitivity of contraction-response curves to shift to the right and/or depress the maximum contraction to a range of agents is probably dependent on the efficiency of receptor-transduction coupling. For example, the concentration-contraction curve for methoxamine was right-shifted 10-fold with a near normal maximum in the presence of cannabidiol 0.3-1 µM suggesting α1-adrenoceptor reserve before the maximum response collapsed in the presence of cannabidiol 3 µM. In contrast, for most other contraction agents, generally the concentration-response curves were markedly depressed at 1 or 3 µM cannabidiol. It is important to acknowledge that our 30 min incubation of the small arteries with cannabidiol 0.1-3 µM allows a longer equilibration time than applying cumulative concentrations of cannabidiol to a precontracted artery (see BaranowskaKuczko et al., 2020). Our results clearly show that cannabidiol pretreated for 30 min does significantly right-shift and/or attenuate the concentration-response curves to a range of constrictor agents. At cannabidiol 1 µM, the subsequent concentration-response curves to endothelin-1 and U46619 were hardly affected but at cannabidiol 3 µM these concentration-response curves were significantly depressed. In comparison, the concentration-response curve to methoxamine and 5-HT were very sensitive to cannabidiol at just 1 µM. However, in the results from Baranowska-Kuczko et al. ((2020) also in rat small mesenteric arteries, they found that 1 µM cannabidiol pretreatment had no effect on subsequent concentration-response curves to phenylephrine in WKY, SHR, or DOCA salttreated rats. They did not report the effect of any higher concentration of cannabidiol in this protocol. Further, they reported that cannabidiol added from 0.1-30 µM on submaximally contracted arteries with phenylephrine could completely inhibit the contraction. We contend that in our present cannabidiol pretreatment protocol, there is the distinct advantage of a 30 min equilibration before completing the contractile agent concentration-response curve. It is important to note the rat mesenteric small artery is very sensitive to just a 3-fold increase in cannabidiol. For example, the concentration-response curve to U46619 was unaffected by pretreatment with 1 µM cannabidiol, but completely inhibited with 3 µM cannabidiol. Similarly, the 5-HT concentration-response curve was unaffected by 0.3 µM cannabidiol but completely inhibited by just 1 µM cannabidiol pretreatment (Fig. 1C and E).
In the vasculature the effects of cannabidiol have been suggested to be mediated by the endothelium, superoxide dismutase, cyclooxygenase, prostanoid EP4, NOS, calciumactivated potassium channels (KCa), CB1-, CB2-, TRPV1- and PPARγ receptors (BaranowskaKuczko et al., 2020; O’Sullivan et al., 2009; Stanley et al., 2015; Wheal et al., 2014; Wheal et al., 2017). In Zucker diabetic fatty (ZDF) rats, chronic in vivo treatment with cannabidiol enhanced vasorelaxation to acetylcholine of isolated small mesenteric arteries (Wheal et al., 2017). The effects of cannabidiol were mediated by cyclooxygenase and NOS.
Cannabidiol had no effect on arteries from corresponding non-diabetic rats. Another study suggested that the effects of acute administration of cannabidiol in small mesenteric arteries isolated from spontaneously hypertensive rats, DOCA-salt hypertensive rats or normotensive controls were mediated by CB1 receptors, in addition to the involvement of CB2 receptors and the endothelium to a varying degree (Baranowska-Kuczko et al., 2020). In our study, neither denuding the endothelium (Supplementary Fig. 1) nor inhibition of cyclooxygenase, NOS, CB1-, CB2-receptors or PPARγ completely reversed the effects of cannabidiol on methoxamine and 5-HT contractions. Cannabidiol-induced inhibition of contractions was partly reversed by the CGRP receptor inhibitor olcegepant in the small mesenteric and saphenous arteries. The co-incubation of arteries with L-NAME, indomethacin and olcegepant significantly reversed most of the effects of cannabidiol on methoxamine and 5-HT contractions. While CGRP may play a role in mediating the effects of cannabidiol, we cannot be sure that cannabidiol is targeting the release of CGRP from perivascular sensory neurons, as the inhibition of prejunctional TRPV1 receptors had no effect on the contractions. The reversal of most of the effects of cannabidiol in the presence of both L-NAME and olcegepant may be explained by the involvement of both NO dependent- and independent-pathways in the effects of CGRP (Kumar et al., 2019). The dependence on NO varies between different vascular beds, whereas in rat mesenteric artery smooth muscle cells, CGRP-induced relaxation was independent of the endothelium (McNeish et al., 2012), which may explain the inability of L-NAME and endothelial denudation alone to fully reverse the effects of cannabidiol. We confirmed that exogenous CGRP, similarly to cannabidiol, caused a concentration-dependent inhibition of contraction to methoxamine, where the lower concentrations of CGRP (1-10 nM) dextrally shifted the contraction curves, while the higher concentration (100 nM) caused both a significant dextral shift and attenuation of Rmax. This similar pattern of cannabidiol and exogenous CGRP on methoxamine curves is compelling.
O-1918, a cannabidiol analogue, has been claimed to inhibit an endothelial cannabinoid receptor (CBe), which has been suggested to mediate the vasodilatory effects of various cannabinoids (Offertaler et al., 2003). In our study, O-1918 reversed the effects of cannabidiol on contractions to 5-HT, while having no effect on methoxamine-contracted arteries. The involvement of a potential CBe receptor should be interpreted with caution as it is known today that O-1918 has several endothelium-independent off-targets at concentrations (3-10 µM), which are the concentrations normally used to verify the involvement of CBe receptors in the effects of cannabinoids (Bondarenko, 2014). In our study, we observed that the incubation of small mesenteric arteries with the higher concentration of O-1918 (10 µM) led to spontaneous spike-like contractions of small mesenteric arteries prior to any addition of contractile agonists (Supplementary Fig. 2).
Cannabidiol (1-3 µM) caused concentration-dependent inhibition of calcium entry through voltage-operated calcium channels in arteries stimulated with both methoxamine (10 µM) and KCl (80 mM), in the absence of extracellular calcium. In addition, cannabidiol blocked the L-type voltage-operated calcium channel agonist Bay K8644 from contracting arteries. Abnormal-cannabidiol, a synthetic cannabidiol analogue, has also been reported to mediate some of its effects by inhibiting the entry of calcium through voltage-operated calcium channels (Ho and Hiley, 2003). While CGRP potently inhibited calcium contractions of arteries in the absence of extracellular calcium, it is unlikely that CGRP is involved in the calcium inhibitory effects of cannabidiol, as olcegepant did not reverse the effects of cannabidiol on calcium contractions (Supplementary Fig. 3).

4.2 Large arteries and non-vascular tissues

Our data show that cannabidiol and exogenous CGRP inhibit the contraction of small arteries to methoxamine. In contrast, in the large saphenous artery, the weak inhibitory action of cannabidiol was matched by the weak action of exogenous CGRP (100 nM). The failure of therapeutic concentrations (1-3 µM) of cannabidiol to alter contraction agents in large arteries is in stark contrast to the resistance arteries. Moreover, the 10-100 times higher concentrations of cannabidiol that were almost without effect on non-vascular tissues highlight a very targeted action in small arteries.
In conclusion, we have demonstrated a sensitive non-specific action of therapeutic concentrations of cannabidiol in isolated small resistance arteries. This may contribute to local vascular bed vasodilatation possibly due to local action of CGRP and L-type voltageoperated calcium channel inhibition. This is in stark contrast to the lack of inhibitory action

References

Abood, M., Alexander, S.P., Barth, F., Bonner, T.I., Bradshaw, H., Cabral, G., Casellas, P., Cravatt, B.F., Devane, W.A., Di Marzo, V., Elphick, M.R., Felder, C.C., Greasley, P., Herkenham, M., Howlett, A.C., Kunos, G., Mackie, K., Mechoulam, R., Pertwee, R.G., Ross, R.A., 2019. Cannabinoid receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database. IUPHAR/BPS Guide to Pharmacology CITE 2019(4).
Angus, J.A., Hughes, R.J.A., Wright, C.E., 2017. Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism. Pharmacol Res Perspect 5.
Angus, J.A., Wright, C.E., 2000. Techniques to study the pharmacodynamics of isolated large and small blood vessels. J Pharmacol Toxicol Methods 44, 395-407.
Baranowska-Kuczko, M., Kozlowska, H., Kloza, M., Sadowska, O., Kozlowski, M., Kusaczuk, M., Kasacka, I., Malinowska, B., 2020. Vasodilatory effects of cannabidiol in human pulmonary and rat small mesenteric arteries: modification by hypertension and the potential pharmacological opportunities. J Hypertens 38, 896-911.
Bondarenko, A.I., 2014. Endothelial atypical cannabinoid receptor: do we have enough evidence? Br J Pharmacol 171, 5573-5588.
Christopoulos, A., Coles, P., Lay, L., Lew, M.J., Angus, J.A., 2001. Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens. Br J Pharmacol 132, 1281-1291.
Devinsky, O., Cross, J.H., Laux, L., Marsh, E., Miller, I., Nabbout, R., Scheffer, I.E., Thiele, E.A., Wright, S., Cannabidiol in Dravet Syndrome Study, G., 2017. Trial of cannabidiol for drug-resistant seizures in the Dravet syndrome. N Engl J Med 376, 2011-2020.
Devinsky, O., Patel, A.D., Cross, J.H., Villanueva, V., Wirrell, E.C., Privitera, M., Greenwood, S.M., Roberts, C., Checketts, D., VanLandingham, K.E., Zuberi, S.M., Group, G.S., 2018a. Effect of cannabidiol on drop seizures in the Lennox-Gastaut syndrome. N Engl J Med 378, 1888-1897.
Devinsky, O., Patel, A.D., Thiele, E.A., Wong, M.H., Appleton, R., Harden, C.L., Greenwood, S., Morrison, G., Sommerville, K., Group, G.P.A.S., 2018b. Randomized, dose-ranging safety trial of cannabidiol in Dravet syndrome. Neurology 90, e1204-e1211.
Geffrey, A.L., Pollack, S.F., Bruno, P.L., Thiele, E.A., 2015. Drug-drug interaction between clobazam and cannabidiol in children with refractory epilepsy. Epilepsia 56, 1246-1251.
Ho, W.S., Hiley, C.R., 2003. Vasodilator actions of abnormal-cannabidiol in rat isolated small mesenteric artery. Br J Pharmacol 138, 1320-1332.
Jadoon, K.A., Tan, G.D., O’Sullivan, S.E., 2017. A single dose of cannabidiol reduces blood pressure in healthy volunteers in a randomized crossover study. JCI Insight 2, e93760.
Kassab, S.E., Khedr, M.A., Ali, H.I., Abdalla, M.M., 2017. Discovery of new indomethacinbased analogs with potentially selective cyclooxygenase-2 inhibition and observed diminishing to PGE2 activities. Eur J Med Chem 141, 306-321.
Kumar, A., Potts, J.D., DiPette, D.J., 2019. Protective role of α-calcitonin gene-related peptide in cardiovascular diseases. Front Physiol 10, 821.
Leesnitzer, L.M., Parks, D.J., Bledsoe, R.K., Cobb, J.E., Collins, J.L., Consler, T.G., Davis, R.G., Hull-Ryde, E.A., Lenhard, J.M., Patel, L., Plunket, K.D., Shenk, J.L., Stimmel, J.B., Therapontos, C., Willson, T.M., Blanchard, S.G., 2002. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41, 6640-6650.
McHugh, D., Hu, S.S., Rimmerman, N., Juknat, A., Vogel, Z., Walker, J.M., Bradshaw, H.B., 2010. N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci 11, 44.
McNeish, A., Roux, B., Aylett, S.-B., Van Den Brink, A., Cottrell, G., 2012. Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries. Br J Pharmacol 167, 1679-1690.
McPartland, J.M., Duncan, M., Di Marzo, V., Pertwee, R.G., 2015. Are cannabidiol and Delta(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review. Br J Pharmacol 172, 737-753.
Mechoulam, R., Parker, L.A., Gallily, R., 2002. Cannabidiol: an overview of some pharmacological aspects. J Clin Pharmacol 42, 11S-19S.
O’Sullivan, S.E., Sun, Y., Bennett, A.J., Randall, M.D., Kendall, D.A., 2009. Time-dependent vascular actions of cannabidiol in the rat aorta. Eur J Pharmacol 612, 61-68.
Offertaler, L., Mo, F.M., Batkai, S., Liu, J., Begg, M., Razdan, R.K., Martin, B.R., Bukoski, R.D., Kunos, G., 2003. Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor. Mol Pharmacol 63, 699-705.
Randall, M.D., Kendall, D.A., O’Sullivan, S., 2004. The complexities of the cardiovascular actions of cannabinoids. Br J Pharmacol 142, 20-26.
Sheykhzade, M., Amandi, N., Pla, M.V., Abdolalizadeh, B., Sams, A., Warfvinge, K., Edvinsson, L., Pickering, D.S., 2017. Binding and functional pharmacological characteristics of gepant-type antagonists in rat brain and mesenteric arteries. Vascul Pharmacol 90, 36-
Stanley, C.P., Hind, W.H., Tufarelli, C., O’Sullivan, S.E., 2015. Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation. Cardiovasc Res 107, 568-578.
Stanley, C.P., Wheal, A.J., Randall, M.D., O’Sullivan, S.E., 2013. Cannabinoids alter endothelial function in the Zucker rat model of type 2 diabetes. Eur J Pharmacol 720, 376382.
Voets, T., Droogmans, G., Wissenbach, U., Janssens, A., Flockerzi, V., Nilius, B., 2004. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430, 748-754.
Wheal, A.J., Cipriano, M., Fowler, C.J., Randall, M.D., O’Sullivan, S.E., 2014. Cannabidiol improves vasorelaxation in Zucker diabetic fatty rats through cyclooxygenase activation. J Pharmacol Exp Ther 351, 457-466.
Wheal, A.J., Jadoon, K., Randall, M.D., O’Sullivan, S.E., 2017. In vivo cannabidiol treatment improves endothelium-dependent vasorelaxation in mesenteric arteries of Zucker diabetic fatty rats. Front Pharmacol 8, 248.