Further characterisation of the time-dependent vascular

Transcription

1 JPET Fast Forward. Published on December 13, 25 as DOI:1.1124/jpet JPET #95828 Further characterisation of the time-dependent vascular effects of 9 -tetrahydrocannabinol (THC). Saoirse E O Sullivan, David A Kendall and Michael D Randall School of Biomedical Sciences, E Floor, Queen s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK (SOS, DAK & MDR). Copyright 25 by the American Society for Pharmacology and Experimental Therapeutics.

2 Running title: Time-dependent vascular effects of THC Corresponding author: Saoirse E. O Sullivan School of Biomedical Sciences, E Floor, Queen s Medical Centre, University of Nottingham, NG7 2UH, UK. Phone Fax Saoirse.o sullivan@nottingham.ac.uk Number of pages 3 Number tables 2 Number of Fig.s 1 Number of references 27 Number of words in abstract 246 Number of words in introduction 57 Number of words in discussion 1617 Abbreviations : CB, cannabinoid; ChTX, charybdotoxin; DETCA, diethyldithiocarbamate; EDHF, endothelium-derived hyperpolarizing factor; H 2 O 2, hydrogen peroxide production; G3, third order branch of the superior mesenteric artery; G, the superior mesenteric artery; L- NAME, N G -nitro-l-arginine methyl ester; NO, nitric oxide; PPARγ, peroxisome proliferatoractivated receptor gamma; PPRE, peroxisome proliferated activated response element; RXR, retinoid X receptor; SOD, superoxide dismutase; THC, 9 -tetrahydrocannabinol. Section assignment : Cardiovascular 2

3 ABSTRACT We have previously shown that over time (2 h), the active ingredient of cannabis, 9 - tetrahydrocannabinol (THC) produces peroxisome proliferator-activated receptor gamma (PPARγ)-mediated vasorelaxation of conduit arteries. We have now investigated whether incubation with THC affects agonist-stimulated contractile (methoxamine) and endotheliumdependent vasorelaxant (acetylcholine) responses in the rat superior mesenteric artery (G) and aorta by myography. We have also investigated whether similar responses are observed in isolated resistance (G3) vessels of the mesenteric bed. In both the aorta and G, incubation with THC (1 µm) for 2 h, but not 1 min, significantly attenuated the contractile responses to methoxamine. This effect of THC was abolished in the presence of the enzyme catalase, which breaks down hydrogen peroxide (H 2 O 2 ), and was reduced in the presence of the superoxide dismutase (SOD) inhibitor, DETCA, but was not PPARγ-mediated. THC also inhibited calcium influx in a H 2 O 2 -dependent manner. In G, but not the aorta, incubation with THC (1 µm, 2 h) significantly enhanced endothelium-dependent vasorelaxation. This was inhibited by a PPARγ antagonist (GW9662), catalase and DETCA, but not by the NO synthase inhibitor L-NAME. By contrast, in G3, no time-dependent vasorelaxation of precontracted arteries to THC was observed, and incubation with THC led to potentiation of contractile responses and blunting of vasorelaxation to acetylcholine, which appears to involve inhibition of endothelium-derived hyperpolarising factor (EDHF) production, and agonist-stimulated production of EDHF. These data demonstrate further the time-dependent vascular actions of THC, and also highlight the heterogenous effects of THC in different arterial types. 3

4 Introduction In the early 199s, a receptor for the active constituent of marijuana, 9 -tetrahydrocannabinol (THC) was identified, followed by the discovery of an endogenous ligand for this receptor, anandamide (Devane et al., 1992). The first in vitro cardiovascular studies showed that both anandamide and THC were capable of relaxing rabbit cerebral arterioles (Ellis et al., 1995), initiating a wealth of research into the cardiovascular effects of anandamide. It has now been widely shown that anandamide causes vasorelaxation through a number of mechanisms involving the endothelium, sensory nerves, and modulation of ion channels (for review see Randall et al., 24). By contrast, the effects of THC on blood vessels have been comparatively neglected; however it has been shown that THC causes acute vasorelaxation of various arterial preparations through a variety of mechanisms involving prostanoids (Ellis et al., 1995; O Sullivan et al., 25a), the endothelium (Fleming et al. 1999; O Sullivan et al., 25a), sensory nerves (Zygmunt et al., 22), and inhibition of calcium channels in combination with activation of potassium channels (O Sullivan et al., 25a). Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear receptors of which there are three isoforms: α, δ and γ (Ferre, 24). When activated, PPARs translocate to the nucleus where they heterodimerise with the retinoid X receptor and bind to DNA sequences leading to the transcription of responsive genes (for review see Bishop- Bailey, 2). PPARγ was traditionally thought to be involved mainly in adipogenesis; however, it has recently become clear that PPARγ is widely expressed with a range of physiological roles (Braissant et al., 1996; Bishop-Bailey, 2). PPARγ agonists are used in the management of Type 2 Diabetes (Ferre, 24; Rangwala & Lazar, 24), but have also been shown to have additional positive cardiovascular effects (Bishop-Bailey, 2; Hsueh & 4

5 Bruemmer, 24). These include in vitro evidence of increased availability of nitric oxide (NO), and in vivo reductions in blood pressure, anti-inflammatory actions, and attenuation of atherosclerosis after PPARγ administration. We have also shown that the PPARγ ligand rosiglitazone causes time-dependent, protein synthesis-dependent vasorelaxation of the rat aorta (Cunnane et al., 24). PPARs are relatively promiscuous and are activated by a number of natural and synthetic ligands. Recent evidence has shown that various cannabinoid compounds and their and metabolites activate PPARs. The endocannabinoid, oleylethanolamide, regulates feeding through activation of PPARα (Fu et al., 23), and metabolism of another endocannabinoid, 2-arachidonoylglycerol (2-AG), causes release of 15-hydroxyeicosatetraenoic acid glyceryl ester, a PPARα agonist (Kozak et al., 22). Similarly, the endocannabinoid, palmitolyethanolamide, has anti-inflammatory properties mediated by PPARα (Lo Verme et al., 25). It has also been reported that ajulemic acid, a 9 -tetrahydrocannabinol (THC) metabolite analogue, binds to PPARγ with potential anti-inflammatory actions (Liu et al., 23). On this basis, we previously investigated whether THC itself is a PPARγ ligand, causing time-dependent vasorelaxant effects via PPARγ activation, and demonstrated that THC is indeed a PPARγ ligand, activation of which increases superoxide dismutase activity, leading to time-dependent vasorelaxation through increased bioavailability of nitric oxide and hydrogen peroxide production (O Sullivan et al., 25b). The aims of the present study were therefore to investigate further the time-dependent effects of THC in conduit arteries by studying the ability of THC to modulate agoniststimulated vasorelaxation and vasoconstriction. Since our previous work investigated only larger conduit arteries (the superior mesenteric artery and aorta), and we have now also investigated the time-dependent effects of THC in isolated mesenteric resistance arteries. 5

6 Material and Methods Blood vessel preparation. Male Wistar rats (25-35g) were stunned by a blow to the back of the head and killed by cervical dislocation. The aorta, superior mesenteric artery and mesenteric arterial bed were removed rapidly and placed into ice-cold Krebs-Henseleit buffer (composition, mm: NaCl 118, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25, CaCl 2 2, D- glucose 1). From the mesenteric arterial bed, 2 mm segments of third-order branches of the superior mesenteric artery (G3) were dissected free of adherent connective and adipose tissue. G3 vessels were mounted on fine tungsten wires (4 µm diameter) on a Mulvany Halpern myograph (Myo-Interface Model 41A, Danish Myo Technology, Denmark) (Mulvany & Halpern, 1977). The superior mesenteric artery (G; 3-4 mm in length) was also cleaned of adherent tissue and was mounted on fixed segment support pins using the Multi Myograph system (Model 61M, Danish Myo Technology, Denmark). The aortae were dissected free of adherent connective and adipose tissue and cut into rings 3-4 mm long, and also mounted on fixed segment support pins using the Multi Myograph system. In all vessels, tension was measured and was recorded on a MacLab 4e recording system (ADInstruments, Oxfordshire, UK). Once mounted, vessels were kept at 37 C in Krebs-Henseleit buffer and gassed with 5% CO 2 in O 2. The mesenteric vessels were stretched to an optimal passive tension of g and the aorta was stretched to g. All vessels were allowed to equilibrate and the contractile integrity of each was initially tested by its ability to contract to 6 mm KCl by at least g. For each experiment, vehicle- and THC-treated experiments were performed in adjacent segments of the same artery. Effects of THC on contractile responses. In G3, G and the aorta, the effects of THC on contractile function were examined by constructing concentration-response curves to methoxamine in adjacent segments of artery 2 h after adding either THC (1 µm) or vehicle 6

7 (5 µl ethanol) to the organ baths. For all arteries, tone was readjusted to g before the addition of methoxamine as over the 2 hr incubation period, there tended to be both increases and decreases in tone, although it was not observed that there was a consistent change in tone in response to THC from baseline. The effects of long-term incubation with THC was compared to the acute effects of THC (1 µm) where methoxamine concentration-response curves were performed 1 min after addition of THC/vehicle. In each case, there was no difference between adjacent segments in their ability to contract to a high potassium solution prior to the incubation periods, therefore the contractile potential of each segment should be similar. The possible contribution of alterations in nitric oxide (NO) were established by performing experiments in the presence of the nitric oxide synthase (NOS) inhibitor N G -nitro- L-arginine methyl ester (L-NAME, 3 µm), which was added to the Krebs solution and present throughout the entire experiment. In G3 vessels, the role of prostanoids in the response to THC were assessed by performing experiments in the presence of the cyclooxygenase inhibitor, indomethacin (1 µm). Again, indomethacin was added to the Krebs solution and present throughout the entire experiment A role for hydrogen peroxide production was investigated by performing experiments in the presence of the catalase (25 u per ml), which metabolises H 2 O 2 into water and oxygen and thus terminates the biological actions of H 2 O 2. Catalase was added to the Krebs solution and present throughout the entire experiment. To test whether changes in superoxide dismutase (SOD) activity contributes to the vascular effects of THC, vessels were pre-treated with the SOD inhibitor diethyldithiocarbamate (DETCA, 3 mm, added 3 min prior to the addition of THC/vehicle, Paisley & Martin, 1996). A potential role for PPARγ activation was investigated using the PPARγ antagonist, GW9662 (1 µm, Leesnitzer et al., 22). In these experiments, GW9662 7

8 was added to the organ baths 1 min before the 2 h THC incubation. In G3 vessels, to establish a role for changes in endothelium-derived hyperpolarising factor (EDHF) production, some experiments were performed in the presence of L-NAME and indomethacin in combination with charybdotoxin to block large calcium-activated K + channels and voltage sensitive K + channels (ChTX, 1 nm) and apamin to block small calcium-activated K + channels (5 nm) as this combination inhibits EDHF responses. In some experiments, the effects of ChTX and apamin alone in the buffer were tested. In all cases, the potassium channels blockers were added to the organ baths 1 min before the 2 h THC incubation. Effects of THC on agonist-stimulated vasorelaxant responses. In G3, G and the aorta, the effects of long-term incubation (2 h) with THC (1 µm) on agonist-stimulated vasorelaxation were examined by performing concentration-response curves to acetylcholine in vehicle- and THC-treated adjacent segments of artery pre-contracted by the methoxamine concentration response curve as described above (maximum dose 1 µm methoxamine). A potential role for NO (L-NAME, 3 µm), hydrogen peroxide (catalase, 25 u per ml), SOD activity (DETCA, 3 mm) or EDHF inhibition (L-NAME 3 µμ, indomethacin 1 µμ, ChTX, 1 nm and apamin 5 nm) were examined in subsequent experiments. Effects of THC on calcium channels. To investigate the effects of THC on calcium influx, concentration-response curves to calcium chloride (CaCl 2, 1 µm to 1 mm) were performed in vehicle controls and in the presence of THC (1 µm, 2h). The vessels were first allowed to equilibrate in calcium-free Krebs, and were then bathed in calcium-free, high potassium (8 mm) Krebs solution. After 2 h, a concentration-response curve to CaCl 2 was constructed. Some experiments were also performed in the presence of catalase (25 u per ml) to inhibit hydrogen peroxide activity. 8

9 Statistical analysis. The concentration of vasorelaxant giving the half-maximal response (EC 5 ) and maximal responses (R max ) were obtained from the concentrationresponse curve fitted to a sigmoidal logistic equation with the minimum vasorelaxation set to zero using the GraphPad Prism package (Tep-areenan et al., 23). Maximal responses (R max ) and pec 5 (negative logarithm of the EC 5 ) values are expressed as mean ± SEM. The number of animals in each group is represented by n. In each protocol, the difference between THC-treated and vehicle-treated vessels were analysed by paired Student s t-test. Other data were compared, as appropriate, by Student s t-test or by analysis of variance (ANOVA) with statistical significance between manipulations and controls determined by Dunnett s post-hoc test Drugs. All drugs were supplied by Sigma Chemical Co. (Poole, UK) except where stated. GW9662 was obtained from Tocris (UK). Acetylcholine, methoxamine and DETCA were dissolved in distilled water. L-NAME and catalase were dissolved into the Krebs- Henseleit solution. Indomethacin was dissolved first in 1 µl ethanol and then dissolved into the Krebs-Henseleit solution. THC was dissolved in ethanol at 1 mm with further dilutions made in distilled water. GW9662 was dissolved in DMSO to 1 mm, with further dilutions made in distilled water. 9

10 Results After 2 h incubation with THC (1 µm), the contractile potency of methoxamine was significantly enhanced in isolated resistance vessels (G3) of the rat mesentery (vehicle pec 5 = 5.5 ± 5 cf THC pec 5 = 5.96 ± 8, n=6, P<1, Fig. 1a). By contrast, in both the superior mesenteric artery (n=8, Fig. 1c, Table 1) and the aorta (n=1, Fig. 1e, Table 1), after 2 h incubation with THC (1 µm), the maximal contractile response to methoxamine was significantly inhibited. The vasorelaxant response to acetylcholine in G3 was significantly inhibited after 2 h incubation with THC (1 µm) (vehicle pec 5 = 7.93 ±.15, cf THC pec 5 = 7.41 ±.12, n=5, P<5, Fig. 1b), while the vasorelaxant response to acetylcholine was significantly enhanced in the superior mesenteric artery after 2 h incubation with THC (1 µm) (n=8, Fig. 7a, Table 2). The endothelium-dependent vasorelaxant responses to acetylcholine in the aorta were not affected by the presence of THC (see Fig. 1f). Mechanisms of time-dependent effects of THC in the isolated aorta (see Table 1,2) The inhibitory effects of THC on contractile responses to methoxamine were not affected if vessels were washed out after the 2 h incubation period (THC 2 h R max = 1.74 ±.18 g cf THC 2 h & washout R max = 1.97 ±.29 g, n=5, Fig.. 2a). Additionally, short-term incubation with THC (1 µm, 1 min) did not significantly alter the subsequent contractile responses to methoxamine (n=7, Fig. 2b). In the presence of L-NAME, THC continued to significantly inhibit the contractile responses to methoxamine (n=5, Fig. 2c). The inhibitory effects of THC on methoxamine-induced responses in the aorta were also not affected by the PPARγ antagonist GW9662 (1 µm, n=6, Fig. 2d). However, in the presence of the catalase, there was no difference in the contractile response between vehicle- and THC-treated vessels (n=8, 1

11 Fig. 2e). In the presence of the SOD inhibitor, DETCA (3 mm), THC inhibited the effects of methoxamine compared with vehicle-treated vessels (n=7, Fig. 2f). However, the percentage inhibition of the methoxamine response caused by THC in the presence of DETCA was significantly less than in the control situation (control 49.8 ± 5.4 % inhibition cf DETCA 24.1 ± 5.4 % inhibition, P<5, ANOVA) (% inhibition in the presence L-NAME = 44.1 ± 9.4 %, % inhibition in the presence of GW9662 = 53. ± 7.3 %). The contractile responses to the re-introduction of calcium in a calcium-free, high potassium Krebs -Hensleit solution was significantly reduced after incubation with THC (1 µm 2 h) in the lower concentration range (1 to 3 µm CaCl 2, P<5 (Student s t-test paired analysis, n=9, see Fig. 3a). In the presence of catalase, this effect of THC was reversed such that arteries incubated with THC tended to contract more to the re-introduction of calcium than vehicle-treated vessels (see Fig. 3b). The vasorelaxant effects of acetylcholine were not altered under any conditions in the presence of THC (Figure 4a-f). In summary, 2 h incubation with THC (1 µm) in the aorta leads to the inhibition of contractile responses to methoxamine, which is time-dependent, not washed out, involve hydrogen peroxide and partly involve SOD activity, but do not involve nitric oxide or the PPARγ receptor. THC also blocks calcium channels in the aorta, also involving hydrogen peroxide. THC incubation (2 h) had no effect on subsequent vasorelaxant response to acetylcholine. 11

12 Mechanisms of time-dependent effects of THC in the superior mesenteric artery (see Table 1,2) Short-term incubation with THC (1 min) did not significantly alter the subsequent contractile responses to methoxamine (n=5, Fig. 5b). In the presence of L-NAME, THC significantly reduced the contractile response to methoxamine (n=6, Fig. 5c). However, in the presence, of the hydrogen peroxide inhibiting enzyme catalase, the inhibitory effects of THC on methoxamine-induced responses were absent (n=6, Fig. 5e). In the presence of the SOD inhibitor, DETCA (3 mm), THC inhibited the contractile responses to methoxamine (n=7, Fig. 5f), although this inhibitory effect tended to be smaller than that seen in the control experiments where THC caused around 5 % inhibition of the methoxamine response (in the presence of DETCA, this was about 25 %, see Fig.5a and Fig 5f). The inhibitory effects of THC on methoxamine responses in the superior mesenteric artery were not affected by the PPARγ antagonist GW9662 (n=6, Fig. 5d). The contractile responses to the re-introduction of calcium in a calcium-free, high potassium Krebs -Hensleit solution were significantly reduced in the presence of THC from 1 µm to 3 mm (n=7, see Fig. 6a). The inhibitory effect of THC was completely abolished when similar experiments were performed in the presence of catalase (n=5, see Fig. 6b). After 2 h incubation with THC (1 µm), the maximal vasorelaxant response (Veh R max = 99.2 ± 7.4 % relaxation, THC R max = 13 ± 12 % relaxation, n=8, P<5, Fig. 7a) to acetylcholine was significantly enhanced compared with vehicle-treated vessels, but this was not seen after short-term incubation with THC (1 min, n=5, Fig. 7b). In the presence of L- NAME, although the vasorelaxant response to acetylcholine was greatly reduced, THC continued to enhance the vasorelaxant effect of acetylcholine compared with vehicle-treated vessels (Veh R max = 14.6 ± % relaxation, THC R max = 35.1 ± 2.5 % relaxation, n=6, 12

13 P<1, Fig. 7c). In the presence of L-NAME and catalase combined, the enhancement of vasorelaxation to acetylcholine seen in THC-treated vessels was abolished (THC R max = 8.4 ± % relaxation, n=4, Fig. 7c). In the presence of catalase alone, there was also no difference in the vasorelaxant response to acetylcholine between THC- and vehicle-treated vessels (n=7, Fig. 7e). Nor was there a difference in the vasorelaxant response to acetylcholine between THC- and vehicle-treated vessels in the presence of the SOD inhibitor DETCA (n=6, Fig. 7f) or in the presence of the PPARγ antagonist GW9662 (n=6, Fig. 7d). In summary, incubation with THC (1 µμ) in the superior mesenteric artery causes time-dependent inhibition of contractile response to methoxamine, which involves hydrogen peroxide, calcium channels and SOD activity, but not nitric oxide or the PPARγ receptor. 2 h incubation with THC also leads to an augmentation of the vasorelaxant response to acetylcholine, which was inhibited by hydrogen peroxide inhibition, SOD inhibition and antagonism of the PPARγ receptor. Time-dependent effects of THC in G3 resistance arteries In G3 vessels, THC (1 µm) caused acute vasorelaxation during the first fifteen minutes after administration. However, after 3 min, there was no difference between the THC and vehicle-treated vessels (Fig. 8a), such that unlike that observed in the conduit vessels (O Sullivan et al., 25b), no time-dependent vasorelaxation to THC was seen in G3 vessels (Fig. 8a). Additionally, unlike conduit vessels (O Sullivan et al., 25b), the PPARγ antagonist GW9662 (1 µm) had no effect on vascular responses to THC over time in G3 vessels (Fig. 8b). The effects of THC on methoxamine-induced contraction were not time-dependent, as a similar enhancement of responses was seen after 1 min incubation with THC (vehicle pec 5 = 5.16 ±.15 cf THC pec 5 = 5.6 ± 6, n=7, P<5, Fig. 9a). Similarly, in the 13

14 presence of indomethacin (1 µm), THC enhanced the contractile responses to methoxamine (vehicle pec 5 = 5.75 ± 6 cf THC pec 5 = 6.11 ±.14, n=7, P<5, Fig. 9e). In the presence of L-NAME (3 µm), THC (1 µm, 2h) had no effect on the contractile response to methoxamine (vehicle pec 5 = 6.22 ±.2, cf THC pec 5 = 6.37 ±.21, n=7, Fig.. 9c). In the presence of catalase, there was no difference in the pec 5 of methoxamine between the vehicle-treated and THC-treated vessels (vehicle pec 5 = 5.98 ± 8, cf THC pec 5 = 6.27 ±.17, n=7, Fig. 9b). However, in the presence of catalase, there was an enhancement in the contractile response to lower concentrations of methoxamine in the presence of THC (3 nm methoxamine; veh 8 ± 6 g increase in tension cf THC.65 ±.22 g increase in tension, n=7, P<5, Fig. 9b). Following EDHF inhibition (L-NAME, indomethacin, ChTX & apamin), THC no longer enhanced the contractile effects of methoxamine (vehicle pec 5 = 5.95 ±.2, cf THC pec 5 = 5.93 ±.15, n=6, Fig. 9d). In the presence of ChTX & apamin alone, there was no difference in the potency of methoxamine between vehicle and THC treated vessels (vehicle pec 5 = 6.5 ± 9 cf THC pec 5 = 6.1 ±.24, n=6, Fig. 9f). However, THC did cause a reduction in the maximal response to methoxamine (vehicle R max = 2.32 ±.14 cf THC R max = 4 ±.23 n=6, P<5, Fig. 9f). Incubation with THC (1 µm, 2h) inhibited the vasorelaxant responses to acetylcholine (vehicle pec 5 = 7.96 ±.1 cf THC pec 5 = 7.49 ±.1, n=5, P<1, Fig.1b). In the presence of L-NAME (Fig. 1a), THC greatly attenuated the vasorelaxant responses to acetylcholine. The effect of THC became more prominent with prolonged incubation, as although incubation for 1 min with THC caused some attenuation of the acetylcholine response, this was not significantly different from vehicle-treated vessels (vehicle pec 5 = 7.83 ±.23 cf THC pec 5 = 7.58 ±.3, n=7, Fig. 1b). In the presence of catalase, THC significantly inhibited the maximal vasorelaxant response to acetylcholine 14

15 (vehicle R max = 93.3 ± 3.6 % relaxation cf THC R max = 73.8 ± 4.8 % relaxation, n=6, P<1, Fig. 1c). Under conditions known to inhibit EDHF activity (in the presence of L-NAME and indomethacin in combination with apamin and charybdotoxin), there was no difference in the maximal relaxant response to acetylcholine between vehicle-treated and THC-treated (1 µm, 2 h) (vehicle R max = 3 ± 2.2 % relaxation cf THC R max = 34.4 ± 2.6 % relaxation, n=5, Fig. 1d). The remaining relaxation to acetylcholine obtained under these conditions was inhibited using catalase, again with no difference between vehicle- and THC-treated vessels (vehicle R max = 1.4 ± 1.4 % relaxation cf THC R max = 12. ± % relaxation, n=3, Fig. 1d). In summary, incubation with THC (1 µμ) in resistance vessels of the mesentery increased the potency of methoxamine, which was inhibited by L-NAME and EDHF inhibition, but not by catalase or indomethacin. Furthermore, incubation of vessels with THC reduced the vasorelaxant effects of acetylcholine, which was not affected by L-NAME or catalase, but was blocked when EDHF was inhibited. 15

16 Discussion In the present study, we have examined whether the active ingredient of cannabis, THC, causes time-dependent alterations in agonist-stimulated responses of rat arteries. In these studies we demonstrate for the first time that in conduit arteries, THC blunts the contractile responses to methoxamine, which appears to involve superoxide dismutase, the production of hydrogen peroxide, and calcium channel inhibition. Furthermore, in the superior mesenteric artery, incubation with THC for 2 hours led to augmented endothelium-dependent vasorelaxation to acetylcholine, which was inhibited by PPARγ antagonism and also involved increased stimulated hydrogen peroxide production. By contrast, in isolated mesenteric resistance vessels, THC potentiated the vasoconstrictor effects of methoxamine and inhibited endothelium-dependent vasorelaxation, which appeared to be through the blockade of EDHF activity. These data highlight that the vascular effects of THC are dependent on the vasodilator mechanisms prevalent in a given artery. We have previously demonstrated that THC produces time-dependent vasorelaxation, which was dependent on an intact endothelium, nitric oxide availability, hydrogen peroxide production, and superoxide dismutase (O Sullivan et al., 25b). Importantly, we showed that these effects were mediated by PPARγ activation. These novel data has led to several questions as to whether THC has similar effects in the cardiovascular system as other PPARγ ligands. Our present study firstly aimed to further characterise the in vitro effects of longterm THC exposure to establish whether pre-incubation with THC would lead to changes in agonist-stimulated responses as a consequence of altered protein activity caused by PPARγ activation. We now show that in conduit arteries (the superior mesenteric artery and aorta), 16

17 incubation with THC causes significant blunting of the vasoconstrictor response to methoxamine and that this is a time-dependent event that persists after washout. In both vessels, the effects of THC were not affected by the presence of the nitric oxide synthase inhibitor L-NAME, and were therefore not due to changes in nitric oxide production. However, in the presence of catalase, which metabolises H 2 O 2 into water and oxygen and thus terminates the biological actions of H 2 O 2, the effects of THC were inhibited, suggesting that THC inhibits contractile responses through increased H 2 O 2 production. Superoxide dismutase (SOD) catalyses the conversion of superoxides to H 2 O 2, so we investigated the effects of THC on methoxamine-induced contractile responses in the presence of a SOD inhibitor, DETCA. We found that DETCA reduced the inhibitory effects of THC, indicating that THC increases H 2 O 2 production by enhancing SOD activity. We also showed that preincubation with THC significantly reduced the vasoconstrictor responses to calcium reintroduction in a calcium-free high-potassium solution, suggesting that blockade of calcium entry by THC may also contribute to the blunting of methoxamine-induced responses in both the aorta and superior mesenteric artery. Interestingly, this response to THC was inhibited in the presence of catalase, implicating that blockade of calcium channels by THC involves H 2 O 2 production. In support of these data, it has been previously shown that incubation with H 2 O 2 inhibits agonist-stimulated contractions, although the underlying mechanisms for this remained unclear (Iesaki et al., 1994). Additionally, a role for Ca 2+ channel blockade has been implicated in the vasorelaxant effects of H 2 O 2 in the rat aorta (Yang et al., 1999). Our data would support this suggestion as we found that the contractile response to calcium reintroduction was increased in the presence of catalase, suggesting that basal H 2 O 2 might play a role in the modulation of calcium channels. To summarise, our data suggest that through an increase in SOD activity, THC stimulates H 2 O 2 production, which leads to calcium channel blockade and subsequent inhibition of contractile responses. 17

18 In the superior mesenteric artery, it was found that incubation with THC (2h) led to a significant augmentation of the endothelium-dependent vasorelaxant responses to acetylcholine. This effect of THC persisted in the presence of L-NAME, and was therefore not due to increased stimulation of NO release, but was inhibited in the presence of catalase and DETCA, again pointing towards a role for increased agonist-stimulated H 2 O 2 release through increased SOD activity. It has been previously shown that some of the endotheliumdependent vasorelaxant effects of acetylcholine are through the release of H 2 O 2 in certain vessels (Matoba et al., 2; Hatoum et al., 25). Thus the difference in results obtained between the aorta and superior mesenteric artery may be explained by the fact that in the aorta, H 2 O 2 does not appear to play a role in the vasorelaxant response to acetylcholine, as indicated by the complete inhibition of responses to acetylcholine in the aorta in the presence of L-NAME. However, in the superior mesenteric artery, there was residual vasorelaxation to acetylcholine in the presence of L-NAME, which was sensitive to catalase, indicating a role for H 2 O 2 in the vasorelaxation to acetylcholine in this vessel. It is important to also note that the augmented responses to acetylcholine induced by THC in the presence of L-NAME were also sensitive to catalase, further confirming that the augmented endothelium-dependent vasorelaxation to acetylcholine caused by THC were through augmented stimulated release of H 2 O 2. Unlike the blunting effect of THC on contractile response to methoxamine in the superior mesenteric artery, the increased endothelium-dependent relaxant responses following THC incubation in the vessel were inhibited by the PPARγ antagonist GW9662, which is in line with our previous finding that time-dependent vasorelaxation to THC is PPARγ-mediated 18

19 (O Sullivan et al., 25b). It has also previously been shown that PPARγ ligand treatment augments/restores vasorelaxant responses to acetylcholine in various models of endothelial dysfunction including diabetes (Kanie et al., 23; Majithiya et al., 25) and hypertension (Ryan et al., 24). Additionally, a recent study has shown that PPARγ ligands (ciglitazone or 15-deoxy-Delta12,14-prostaglandin J2) stimulate both activity and expression of Cu/Zn- SOD in HUVECs (Hwang et al., 25), which is also consistent with the present data. Since the time-dependent, SOD-dependent effects of THC on methoxamine-induced responses were PPARγ-independent, it may be that THC is capable of increasing SOD activity and H 2 O 2 production through both PPARγ-dependent and -independent mechanisms. It is of note that in both vasorelaxant effects of acetylcholine in the presence of GW9662 were reduced in both the aorta and the superior mesenteric artery. Our current knowledge of the pharmacology of GW9662 is incomplete and it is possible that it may have additional pharmacological actions. The acetylcholine response is dependent on several components, including muscarinic receptor activation, nitric oxide synthase activity, potassium channel and gap junctional communication, and it is therefore conceivable that actions of GW9662 at one or more of these sites could reduce the response to acetylcholine. Accordingly, further work is required to define the pharmacological activity of GW9662. We previously reported that THC causes time-dependent, PPARγ mediated vasorelaxation of conduit arteries (O Sullivan et al., 25b). In the present study we have examined whether a similar response to THC is observed in isolated resistance arteries of the mesenteric arterial bed. While THC (1 µm) caused the expected acute vasorelaxation of resistance vessels (O Sullivan et al., 25a), after thirty minutes, there was no difference between the THCtreated and vehicle-treated vessels, and furthermore, PPARγ antagonism had no effect on the 19

20 THC response in resistance vessels at any time point. Thus, unlike in the aorta and superior mesenteric artery (O Sullivan et al., 25b), there is no time-dependent PPARγ-mediated vasorelaxation to THC in resistance mesenteric arteries. Although our data do not point towards a reason for the heterogeneity between vessel types, it might be speculated that this could be due to differences expression and /or function of the PPARγ receptor between tissues. Since G3 vessels did not respond similarly to THC as conduit arteries, it was not surprising to find that incubation with THC had the opposite effect on G3 arteries as on conduit vessels; potentiation of methoxamine-induced contractile responses and significant inhibition of vasorelaxant response to acetylcholine. The potentiation of methoxamine responses in G3 were not time-dependent (i.e. were also observed if THC was incubated for only 1 min), persisted in the presence of indomethacin, but were abolished in the presence of L-NAME or after EDHF inhibition. Collectively, this indicates that in G3, THC inhibits nitric oxide and EDHF basal activity but not prostaglandins, leading to enhanced vasoconstrictor responsiveness. Furthermore, in G3, THC significantly reduced the vasorelaxant responses to acetylcholine. It was clear that THC was specifically inhibiting the fast component of relaxation to acetylcholine, which suggests that THC might be inhibiting the EDHF component of the acetylcholine response, as has been previously shown in rabbit mesenteric arteries (Fleming et al., 1999) and mesenteric arteries (O Sullivan et al., 25a). To confirm this, we performed experiments in the presence of L-NAME to eliminate the nitric oxide contribution to vasorelaxation to acetylcholine and found that the inhibition of vasorelaxation by THC persisted (see Fig.. 1b). However, when EDHF activity was blocked, there was no longer any difference between the vehicle- and THC- treated vessels. Taken together, this suggests that THC is capable of inhibiting both the basal and agoniststimulated production of EDHF in resistance vessels. 2

21 Collectively, our data demonstrate that the effects of THC on endothelium-dependent vasorelaxation are clearly dependent on the predominant endothelium-dependent relaxing factor in a given artery. In the aorta, where nitric oxide is the predominate relaxing factor, THC has no effect on agonist-stimulated vasorelaxation. In the superior mesenteric artery, where hydrogen peroxide production contributes to vasorelaxation, THC enhances agoniststimulated endothelium-dependent vasorelaxation, and finally, in resistance mesenteric arteries, where EDHF is the predominant relaxing factor, THC inhibits endotheliumdependent vasorelaxation. In conclusion, we have now shown further time-dependent vascular action of THC in conduit arteries that are both PPARγ-mediated and independent, but both appear to involve increases in SOD activity leading to increased H 2 O 2 production. Importantly, in some vessels, this leads to THC causing an augmentation of endothelium-dependent vasorelaxation. By contrast, in resistance vessels, THC inhibits both basal and stimulated EDHF activity. These data highlight the heterogeneous effects of THC in different arterial types. Acknowledgements We would like to thank Dr Richard Roberts for the use of his myograph. 21

22 References Bishop-Bailey D (2) Peroxisome proliferator-activated receptors in the cardiovascular system. Br J Pharmacol 129: Cunnane SE, Chan YY and Randall MD (24) Rosiglitazone-induced vasorelaxation in the rat aorta. Proceedings of the British Pharmacological Society at Vol2Issue2abst96P. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A and Mechoulam R (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258: Ellis EF, Moore SF and Willoughby KA (1995) Anandamide and delta 9-THC dilation of cerebral arterioles is blocked by indomethacin. Am J Physiol 269:H Ferre P (24) The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes 53:S43-5. Fleming I, Schermer B, Popp R and Busse R (1999) Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists. Br J Pharmacol 126: Fu J, Gaetani S, Oveisi F, Lo Verme J, Serrano A, Rodriguez De Fonseca F, Rosengarth A, Luecke H, Di Giacomo B, Tarzia G, and Piomelli D (23) Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 425:9-93. Hatoum OA, Binion DG, Miura H, Telford G, Otterson MF, and Gutterman DD (25) Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels. Am J Physiol 288:H Hsueh WA and Bruemmer D (24) Peroxisome proliferator-activated receptor gamma: implications for cardiovascular disease. Hypertension 43:

23 Hwang J, Kleinhenz DJ, Lassegue B, Griendling KK, Dikalov S and Hart CM (25) Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol 288:C Iesaki T, Okada T, Yamaguchi H, and Ochi R (1994) Inhibition of vasoactive amine induced contractions of vascular smooth muscle by hydrogen peroxide in rabbit aorta. Cardiovasc Res 28: Kanie N, Matsumoto T, Kobayashi T, and Kamata K. (23) Relationship between peroxisome proliferator-activated receptors (PPAR alpha and PPAR gamma) and endothelium-dependent relaxation in streptozotocin-induced diabetic rats. Br J Pharmacol 14: Kozak KR, Crews BC, Morrow JD, Wang LH, Ma YH, Weinander R, Jakobsson PJ and Marnett LJ (22) 15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist. J Biol Chem 277: Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull- Ryde EA, Lenhard JM, Patel L, Plunket KD, Shenk JL, Stimmel JB, Therapontos C, Willson TM, and Blanchard SG (22) Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41: Liu J, Li H, Burstein SH, Zurier RB and Chen JD (23) Activation and binding of peroxisome proliferator-activated receptor gamma by synthetic cannabinoid ajulemic acid. Mol Pharmacol 63: Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A, and Piomelli D (25) The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the antiinflammatory actions of palmitoylethanolamide. Mol Pharmacol 67:

24 Majithiya JB, Paramar AN, and Balaraman R (25) Pioglitazone, a PPARgamma agonist, restores endothelial function in aorta of streptozotocin-induced diabetic rats. Cardiovasc Res 66: Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, and Takeshita A (2) Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest 16: Mulvany MJ and Halpern W (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41: O'Sullivan SE, Kendall DA, and Randall MD (25a) The vascular effects of 9 - tetrahydrocannabinol (THC) and its interactions with the endocannabinoid anandamide. Br J Pharmacol. 145: O'Sullivan SE, Kendall DA, and Randall MD (25b) Novel time-dependent vascular actions of 9 -tetrahydrocannabinol (THC) mediated by peroxisome proliferator-activated receptor gamma (PPARγ) 337: Paisley K, and Martin W (1996) Blockade of nitrergic transmission by hydroquinone, hydroxocobalamin and carboxy-ptio in bovine retractor penis: role of superoxide anion. Br J Pharmacol 117: Randall MD, O Sullivan SE, and Kendall DA (24) The complexities of the cardiovascular actions of cannabinoids. Br J Pharmacol 142:2-26. Rangwala SM and Lazar MA (24) Peroxisome proliferator-activated receptor gamma in diabetes and metabolism. Trends Pharmacol Sci 25: Ryan MJ, Didion SP, Mathur S, Faraci FM, and Sigmund CD. (24) PPAR(gamma) agonist rosiglitazone improves vascular function and lowers blood pressure in hypertensive transgenic mice. Hypertension 43:

25 Yang Z, Zhang A, Altura BT, and Altura BM (1999) Hydrogen peroxide-induced endothelium-dependent relaxation of rat aorta involvement of Ca 2+ and other cellular metabolites. Gen Pharmacol 33: Zygmunt PM, Andersson DA, and Hogestatt ED (22). Delta 9-tetrahydrocannabinol and cannabinol activate capsaicin-sensitive sensory nerves via a CB1 and CB2 cannabinoid receptor-independent mechanism. J Neurosci 22:

26 Footnotes This study was funded by the British Heart Foundation (PG21/15). 26

27 Figure Legends Fig. 1. The effects of THC (1 µm, 2 h) on vasorelaxant and vasoconstrictor responses in resistance (G3, third order branch of the mesenteric artery) and conduit arteries (the superior mesenteric artery (G) and aorta). Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicletreated adjacent segments of artery (paired Student s t-test). Fig. 2. A, the effects of 1 µm THC (2 h) on the contractile responses to methoxamine after THC was washed out. B, the effects of short-term incubation with 1 µm THC (1 min) on the contractile responses to methoxamine. C, the effects of THC (1 µm, 2 h) on responses to methoxamine in the presence of the NO synthase inhibitor L-NAME (3 µm). D, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of the PPARγ antagonist GW9662 (1 µm, 1 min before THC/vehicle). E, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of catalase (25 u per ml, throughout experiment). F, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of the SOD inhibitor, DETCA (3 mm, 3 min before THC/vehicle). Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicle-treated adjacent segments of artery (paired Student s t-test). Fig. 3. The contractile response to the re-introduction of calcium chloride in a calcium-free, high potassium Krebs solution in the aorta in the absence and presence of THC (1 µm, A). B. The effects of the hydrogen peroxide inhibitor, catalase (25 u per ml, B) on the 27

28 contractile response to the re-introduction of calcium chloride in a calcium-free, high potassium Krebs solution in the aorta. Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicletreated adjacent segments of artery (paired Student s t-test). Fig. 4. Vasorelaxant responses to acetylcholine in the aorta following incubation with THC; THC 2 h (A), THC 1 min (B), in the presence of L-NAME (C), in the presence of GW9662 (D), in the presence of catalase (E) or in the presence of DETCA (F). Data are given as means with error bars representing SEM. Fig. 5. The effects of 1 µm THC after 2 h (A) and after 1 min (B) incubation on the contractile responses to methoxamine in the superior mesenteric artery. C, the effects of THC (1 µm, 2 h) on responses to methoxamine in the presence of the NO synthase inhibitor L- NAME (3 µm, throughout experiment). D, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of the PPARγ antagonist GW9662 (1 µm, 1 min before THC/vehicle). E, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of catalase (25 u per ml, throughout experiment). F, the effects of THC (1 µm, 2 h) on methoxamine-induced responses in the presence of the SOD inhibitor, DETCA (3 mm, 3 min before THC/vehicle). Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicle-treated adjacent segments of artery (paired Student s t-test). Fig. 6. The contractile response to the re-introduction of calcium in a calcium-free, high potassium Krebs solution in the superior mesenteric artery in the presence of THC (1 µm, 2 28

29 h, A). B. The additional presence of the hydrogen peroxide inhibitor, catalase (25 u per ml) on the contractile response to the re-introduction of calcium in a calcium-free, high potassium Krebs solution. Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicle-treated adjacent segments of artery (paired Student s t-test). Fig. 7. The effects of THC (1 µm) on the endothelium-dependent vasorelaxant response to acetylcholine in the superior mesenteric artery after incubation for 2 hours (A) or 1 min (B). The effects of THC (1 µm, 2 h) on endothelium-dependent vasorelaxation to acetylcholine in the presence of L-NAME (3 µm), and L-NAME in combination with catalase (25 units per ml, C). D, the effects of THC (1 µm, 2 h) on vasorelaxation to acetylcholine in the presence of the PPARγ antagonist GW9662 (1 µm). E, the effects of THC (1 µm, 2 h) on endothelium-dependent vasorelaxation to acetylcholine in the presence of catalase (25 units per ml). F, the effects of THCs (1 µm, 2 h) on vasorelaxation to acetylcholine in the presence of the SOD inhibitor, DETCA (3 mm). Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THCand vehicle-treated adjacent segments of artery (paired Student s t-test). Fig. 8. A, the effects of THC (1 µm) on precontracted isolated resistance vessels (G3) over time compared with vehicle treated control vessels. B, the effects of the PPARγ antagonist GW9662 (1 µm) on the residual relaxation to THC (vasorelaxation caused by THC minus vasorelaxation caused by vehicle). Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicletreated adjacent segments of artery (paired Student s t-test). 29

30 Fig. 9. A, the effects of THC (1 µm, 1 min) on the contractile response to methoxamine in isolated resistance arteries of the mesenteric bed (G3). B, the effects of THC (1 µm, 2 h) on the contractile responses to methoxamine in the presence of catalase (25 units per ml). C, the effect of THC (1 µm, 2 h) on the contractile responses to methoxamine in the presence of L-NAME (3 µm). D, the effects of THC (1 µm, 2 h) on the contractile responses to methoxamine in the presence of L-NAME and indomethacin in combination with apamin and charybdotoxin. E, the effects of THC (1 µm, 2 h) on the contractile response to methoxamine in the presence of indomethacin 1 µm). F, the effects of THC (1 µm, 2 h) on the contractile responses to methoxamine in the presence of apamin and charybdotoxin. Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicle-treated adjacent segments of artery (paired Student s t-test). Fig. 1. A, a representative trace showing the effects of THC compared with a vehicletreated segment of the same artery on the vasorelaxant effects of acetylcholine in the presence of the NO synthase inhibitor L-NAME (3 µm). B, the effects of THC (1 µm, 1 min) on the vasorelaxant response to acetylcholine. C, the effects of THC (1 µm, 2 h) on the vasorelaxant response to acetylcholine in the presence of catalase (25 units per ml). D, the effects of THC (1 µm, 2 h) on the vasorelaxant responses to acetylcholine in the presence of L-NAME and indomethacin in combination with apamin and charybdotoxin, and also in the additional presence of catalase. Data are given as means with error bars representing SEM. denotes a significant difference (P<5, P<1) between THC- and vehicle-treated adjacent segments of artery (paired Student s t-test). 3

31 TABLE 1 Time-dependent effects of THC (1 µm) on contractile responses to methoxamine in the aorta and superior mesenteric artery. denotes a significant inhibition of the maximal methoxamine-induced contractile response in THC-treated (1 µm) vessels compared with vehicle-treated (.1 % EtOH) vessels ( <5, P<1). Aorta R max G R max Vehicle (.1% EtOH, 2 h) 2.68 ±.23 g 1.16 ±.15 g THC (1 µm, 2 h) 8 ±.35 g.66 ± 5 g Vehicle (1 min) 1.91 ±.19 g 4 ±.25 g THC (1 µm, 1 min) 1.63 ±.2 g ±.17 g Vehicle & L-NAME (3 µm) 3.55 ±.13 g 1.94 ±.25 g THC & L-NAME (3 µm) 2.11 ±.35 g 1.13 ±.13 g Vehicle & GW9662 (1 µm) 2.2 ±.11 g 1.65 ±.26 g THC & GW9662 (1 µm) 1.16 ±.24 g.98 ±.1 g Vehicle & catalase (25 u/ml) 1.87 ±.23 g 1.81 ±.27 g THC & catalase (25 u/ml) 7 ±.34 g 1.49 ±.17 g Vehicle & DETCA (3 mm) 2.26 ±.2 g 1.67 ±.19 g THC & DETCA (3 mm) 1.87 ±.1 g 1.3 ±.11 g 31

32 TABLE 2 Time-dependent effects of THC (1 µm) on acetylcholine-induced endothelium-dependent vasorelaxant responses in the aorta and superior mesenteric artery. denotes a significant augmentation of the maximal vasorelaxant response in THC-treated vessels (1 µm) compared with vehicle-treated (.1% EtOH) vessels ( <5, P<1). Aorta R max G R max Vehicle (.1% EtOH, 2 h) 59. ± 4.4 % relaxation 99.2 ± 7.4 % relaxation THC (1 µm, 2 h) 66.9 ± 4.6 % relaxation 13 ± 12 % relaxation Vehicle (.1% EtOH, 1 min) 68.9 ± 4.1 % relaxation 89.8 ± 6.3 % relaxation THC (1 µm, 1 min) 62. ± 4.2 % relaxation 93.9 ± 1.1 % relaxation Vehicle & L-NAME (3 µm) 3.29 ± 1.2 % relaxation 14.6 ± % relaxation THC & L-NAME (3 µm) 4.73 ± 2. % relaxation 35.1 ± 2.5 % relaxation Vehicle & GW9662 (1 µm) 45.9 ± 5.9 % relaxation 74.7 ± 6.9 % relaxation THC & GW9662 (1 µm) 44.8 ± 4.4 % relaxation 79.4 ± 8.9 % relaxation Vehicle & catalase (25 u/ml) 62.9 ± 13.9 % relaxation 75. ± 4.6 % relaxation THC & catalase (25 u/ml) 6.4 ± 15.9 % relaxation 83.1 ± 5.7 % relaxation Vehicle & DETCA (3 mm) 56.3 ± 6.9 % relaxation 94.4 ± 11.3 % relaxation THC & DETCA (3 mm) 64.2 ± 4.6 % relaxation 18 ± 16 % relaxation 32