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CARDIOVASCULAR

Further Characterization of the Time-Dependent Vascular Effects of ⁹-Tetrahydrocannabinol

School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom

Received September 19, 2005; accepted December 9, 2005.

	Abstract

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We have previously shown that over time (2 h), the active ingredient of cannabis, ⁹-tetrahydrocannabinol (THC), produces peroxisome proliferator-activated receptor (PPAR) -mediated vasorelaxation of conduit arteries. We have now investigated whether incubation with THC affects agonist-stimulated contractile (methoxamine) and endothelium-dependent vasorelaxant (acetylcholine) responses in the rat superior mesenteric artery (G0) 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 G0, incubation with 10 µM THC for 2 h, but not 10 min, significantly attenuated the contractile responses to methoxamine. This effect of THC was abolished in the presence of the enzyme catalase, which breaks down H₂O₂, and was reduced in the presence of the superoxide dismutase inhibitor diethyldithiocarbamate (DETCA), but it was not PPAR-mediated. THC also inhibited calcium influx in a H₂O₂-dependent manner. In G0, but not the aorta, incubation with 10 µM THC for 2 h significantly enhanced endothelium-dependent vasorelaxation. This was inhibited by a PPAR antagonist, 2-chloro-5-nitro-N-phenylbenzamide (GW9662), catalase, and DETCA, but not by the NO synthase inhibitor N^G-nitro-L-arginine methyl ester. 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 seems to involve inhibition of endothelium-derived hyperpolarizing 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.

Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear receptors of which there are three isoforms: , , and (Ferre, 2004). When activated, PPARs translocate to the nucleus where they heterodimerize with the retinoid X receptor and bind to DNA sequences, leading to the transcription of responsive genes (for review, see Bishop-Bailey, 2000). PPAR was traditionally thought to be involved mainly in adipogenesis; however, it has recently become clear that PPAR is widely expressed with a range of physiological roles (Braissant et al., 1996; Bishop-Bailey, 2000). PPAR agonists are used in the management of type 2 diabetes (Ferre, 2004; Rangwala and Lazar, 2004), but they have also been shown to have additional positive cardiovascular effects (Bishop-Bailey, 2000; Hsueh and Bruemmer, 2004). These include in vitro evidence of increased availability of NO and in vivo reductions in blood pressure, anti-inflammatory actions, and attenuation of atherosclerosis after PPAR administration. We have also shown that the PPAR ligand rosiglitazone causes time-dependent, protein synthesis-dependent vasorelaxation of the rat aorta (Cunnane et al., 2004).

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 PPAR (Fu et al., 2003), and metabolism of another endocannabinoid, 2-arachidonoylglycerol, causes release of 15-hydroxyeicosatetraenoic acid glyceryl ester, a PPAR agonist (Kozak et al., 2002). Similarly, the endocannabinoid palmitolyethanolamide has anti-inflammatory properties mediated by PPAR (Lo Verme et al., 2005). It has also been reported that ajulemic acid, a THC metabolite analog, binds to PPAR with potential anti-inflammatory actions (Liu et al., 2003). On this basis, we previously investigated whether THC itself is a PPAR ligand, causing time-dependent vasorelaxant effects via PPAR activation and demonstrated that THC is indeed a PPAR 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., 2005a).

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 agonist-stimulated vasorelaxation and vasoconstriction. Because our previous work investigated only larger conduit arteries (the superior mesenteric artery and aorta), we have now also investigated the time-dependent effects of THC in isolated mesenteric resistance arteries.

	Materials and Methods

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Blood Vessel Preparation. Male Wistar rats (250–350 g) 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 (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 2 mM CaCl₂, and 10 mM D-glucose). 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 (40 µm in diameter) on a Mulvany-Halpern myograph (Myo-Interface model 410A; Danish Myo Technology, Aarhus, Denmark) (Mulvany and Halpern, 1977). The superior mesenteric artery (G0; 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 610M; Danish Myo Technology). The aortae were dissected free of adherent connective and adipose tissue and cut into rings 3 to 4 mm in length 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% CO₂ in O₂. The mesenteric vessels were stretched to an optimal passive tension of 0.5 g, and the aorta was stretched to 1.0 g. All vessels were allowed to equilibrate, and the contractile integrity of each was initially tested by its ability to contract to 60 mM KCl by at least 0.5 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, G0, 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 10 µM THC or 5 µl of ethanol (vehicle) to the organ baths. For all arteries, tone was readjusted to 0.5 g before the addition of methoxamine because over the 2-h 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 were compared with the acute effects of 10 µM THC where methoxamine concentration-response curves were performed 10 min after addition of THC or vehicle. In each case, there was no difference between adjacent segments in their ability to contract to a high potassium solution before the incubation periods; therefore, the contractile potential of each segment should be similar.

The possible contribution of alterations in NO were established by performing experiments in the presence of the nitric-oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 300 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 was assessed by performing experiments in the presence of the cyclooxygenase inhibitor indomethacin (10 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 (2500 U/ml), which metabolizes H₂O₂ into water and oxygen and thus terminates the biological actions of H₂O₂. Catalase was added to the Krebs' solution and present throughout the entire experiment. To test whether changes in superoxide dismutase (SOD) activity contribute to the vascular effects of THC, vessels were pretreated with the SOD inhibitor diethyldithiocarbamate (DETCA; 3 mM) added 30 min before the addition of THC or vehicle (Paisley and Martin, 1996). A potential role for PPAR activation was investigated using the PPAR antagonist GW9662 (1 mM; Leesnitzer et al., 2002). In these experiments, GW9662 was added to the organ baths 10 min before the 2-h THC incubation. In G3 vessels, to establish a role for changes in endothelium-derived hyperpolarizing factor (EDHF) production, some experiments were performed in the presence of L-NAME and indomethacin in combination with 100 nM charybdotoxin (ChTX) to block large calcium-activated K⁺ channels and voltage-sensitive K⁺ channels and 500 nM apamin to block small calcium-activated K⁺ channels because 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 10 min before the 2-h THC incubation.

Effects of THC on Agonist-Stimulated Vasorelaxant Responses. In G3, G0, and the aorta, the effects of long-term incubation (2 h) with 10 µM THC on agonist-stimulated vasorelaxation were examined by performing concentration-response curves to acetylcholine in vehicle- and THC-treated adjacent segments of artery precontracted by the methoxamine concentration-response curve as described above (maximum dose 100 µM methoxamine). A potential role for NO (L-NAME; 300 µM), hydrogen peroxide (catalase; 2500 U/ml), SOD activity (DETCA; 3 mM), or EDHF inhibition (30 µM L-NAME, 10 µM indomethacin, 100 nM ChTX, and 500 nM apamin) were examined in subsequent experiments.

Effects of THC on Calcium Channels. To investigate the effects of THC on calcium influx, concentration-response curves to CaCl₂ (1 µM–100 mM) were performed in vehicle controls and in the presence of 10 µM THC for 2 h. The vessels were first allowed to equilibrate in calcium-free Krebs' and were then bathed in calcium-free, high potassium (80 mM) Krebs' solution. After 2 h, a concentration-response curve to CaCl₂ was constructed. Some experiments were also performed in the presence of catalase (2500 U/ml) to inhibit hydrogen peroxide activity.

Statistical Analysis. The concentration of vasorelaxant giving the half-maximal response (EC₅₀) and maximal responses (R_max) were obtained from the concentration-response curve fitted to a sigmoidal logistic equation with the minimum vasorelaxation set to zero using the GraphPad Prism package (GraphPad Software Inc., San Diego, CA) (Tep-areenan et al., 2003). R_max and pEC₅₀ (negative logarithm of the EC₅₀) values are expressed as mean ± S.E.M. The number of animals in each group is represented by n. In each protocol, the difference between THC-treated and vehicle-treated vessels was analyzed by paired Student's t test. Other data were compared, as appropriate, by Student's t test or by analysis of variance with statistical significance between manipulations and controls determined by Dunnett's post hoc test.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effects of 10 µM THC for 2 h on vasorelaxant and vasoconstrictor responses in resistance (G3, third order branch of the mesenteric artery) and conduit arteries [the superior mesenteric artery (G0) and aorta]. Data are given as means with error bars representing S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

	Results

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After 2-h incubation with 10 µM THC, the contractile potency of methoxamine was significantly enhanced in isolated resistance vessels (G3) of the rat mesentery (vehicle pEC₅₀ = 5.5 ± 0.05 cf. THC pEC₅₀ = 5.96 ± 0.08; n = 6; P < 0.001; Fig. 1A). By contrast, in both the superior mesenteric artery (n = 8; Fig. 1C; Table 1) and the aorta (n = 10; Fig. 1E; Table 1), after 2-h incubation with 10 µM THC, the maximal contractile response to methoxamine was significantly inhibited. The vasorelaxant response to acetylcholine in G3 was significantly inhibited after 2-h incubation with 10 µM THC (vehicle pEC₅₀ = 7.93 ± 0.15 cf. THC pEC₅₀ = 7.41 ± 0.12; n = 5; P < 0.05; Fig. 1B), whereas the vasorelaxant response to acetylcholine was significantly enhanced in the superior mesenteric artery after 2-h incubation with 10 µM THC (n = 8; Fig. 1D; Table 2). The endothelium-dependent vasorelaxant responses to acetylcholine in the aorta were not affected by the presence of THC (Fig. 1F).

Mechanisms of Time-Dependent Effects of THC in the Isolated Aorta (Tables 1 and 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 ± 0.18 g cf. THC 2-h and washout R_max = 1.97 ± 0.29 g; n = 5; Fig. 2A). Additionally, short-term incubation with 10 µM THC for 10 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 PPAR antagonist GW9662 (1 mM; n = 6; Fig. 2D). However, in the presence of catalase, there was no difference in the contractile response between vehicle- and THC-treated vessels (n = 8; 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 of 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 < 0.05; analysis of variance) (percentage of inhibition in the presence L-NAME = 44.1 ± 9.4, percentage of inhibition in the presence of GW9662 = 53.0 ± 7.3).

View larger version (34K): [in this window] [in a new window]   Fig. 2. A, effects of 10 µM THC (2 h) on the contractile responses to methoxamine after THC was washed out. B, effects of short-term incubation with 10 µM THC (10 min) on the contractile responses to methoxamine. C, effects of THC (10 µM; 2 h) on responses to methoxamine in the presence of the NO synthase inhibitor L-NAME (300 µM). D, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of the PPAR antagonist GW9662 (1 µM; 10 min before THC or vehicle). E, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of catalase (2500 U/ml, throughout experiment). F, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of the SOD inhibitor DETCA (3 mM; 30 min before THC or vehicle). Data are given as means with error bars representing S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

The contractile responses to the reintroduction of calcium in a calcium-free, high potassium Krebs-Henseleit solution was significantly reduced after incubation with 10 µM THC for 2 h in the lower concentration range (10–300 µM CaCl₂; Student's t test paired analysis; P < 0.05; n = 9; 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 reintroduction of calcium than vehicle-treated vessels (Fig. 3B). The vasorelaxant effects of acetylcholine were not altered under any conditions in the presence of THC (Fig. 4, A–F).

View larger version (21K): [in this window] [in a new window]   Fig. 3. A, contractile response to the reintroduction of calcium chloride in a calcium-free, high potassium Krebs' solution in the aorta in the absence and presence of THC (10 µM). B, effects of the hydrogen peroxide inhibitor catalase (2500 U/ml) on the contractile response to the reintroduction of calcium chloride in a calcium-free, high potassium Krebs' solution in the aorta. Data are given as means with error bars representing S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

View larger version (32K): [in this window] [in a new window]   Fig. 4. Vasorelaxant responses to acetylcholine in the aorta following incubation with THC; THC 2 h (A), THC 10 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 S.E.M.

In summary, 2-h incubation with 10 µM THC in the aorta leads to the inhibition of contractile responses to methoxamine, which are time-dependent, not washed out, involve hydrogen peroxide, and partly involve SOD activity, but do not involve nitric oxide or the PPAR receptor. THC also blocks calcium channels in the aorta, also involving hydrogen peroxide. THC incubation for 2 h had no effect on subsequent vasorelaxant response to acetylcholine.

Mechanisms of Time-Dependent Effects of THC in the Superior Mesenteric Artery (Tables 1 and 2). Short-term incubation with THC for 10 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 50% inhibition of the methoxamine response (in the presence of DETCA, this was approximately 25%; see Fig. 5, A and F). The inhibitory effects of THC on methoxamine responses in the superior mesenteric artery were not affected by the PPAR antagonist GW9662 (n = 6; Fig. 5D).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Effects of 10 µM THC after 2-h (A) and after 10-min (B) incubation on the contractile responses to methoxamine in the superior mesenteric artery. C, effects of THC (10 µM; 2 h) on responses to methoxamine in the presence of the NO synthase inhibitor L-NAME (300 µM; throughout experiment). D, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of the PPAR antagonist GW9662 (1 µM; 10 min before THC/vehicle). E, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of catalase (2500 U/ml, throughout experiment). F, effects of THC (10 µM; 2 h) on methoxamine-induced responses in the presence of the SOD inhibitor DETCA (3 mM; 30 min before THC or vehicle). Data are given as means with error bars representing S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

The contractile responses to the reintroduction of calcium in a calcium-free, high potassium Krebs-Hensleit solution were significantly reduced in the presence of THC from 100 µM to 3 µM (n = 7; Fig. 6A). The inhibitory effect of THC was abolished when similar experiments were performed in the presence of catalase (n = 5; Fig. 6B).

View larger version (21K): [in this window] [in a new window]   Fig. 6. A, contractile response to the reintroduction of calcium in a calcium-free, high potassium Krebs' solution in the superior mesenteric artery in the presence of THC (10 µM; 2 h). B, additional presence of the hydrogen peroxide inhibitor catalase (2500 U/ml) on the contractile response to the reintroduction of calcium in a calcium-free, high potassium Krebs' solution. Data are given as means with error bars representing S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

View larger version (35K): [in this window] [in a new window]   Fig. 7. Effects of 10 µM THC on the endothelium-dependent vasorelaxant response to acetylcholine in the superior mesenteric artery after incubation for 2 h (A) or 10 min (B). C, effects of THC (10 mM; 2 h) on endothelium-dependent vasorelaxation to acetylcholine in the presence of 300 µM L-NAME and L-NAME in combination with catalase (2500 U/ml). D, effects of THC (10 µM; 2 h) on vasorelaxation to acetylcholine in the presence of the PPAR antagonist GW9662 (1 µM). E, effects of THC (10 µM; 2 h) on endothelium-dependent vasorelaxation to acetylcholine in the presence of catalase (2500 U/ml). F, effects of THC (10 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 S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

Time-Dependent Effects of THC in G3 Resistance Arteries. In G3 vessels, 10 µM THC caused acute vasorelaxation during the first 15 min after administration. However, after 30 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., 2005a), no time-dependent vasorelaxation to THC was seen in G3 vessels (Fig. 8A). Additionally, unlike conduit vessels (O'Sullivan et al., 2005a), the PPAR antagonist GW9662 (1 µM) had no effect on vascular responses to THC over time in G3 vessels (Fig. 8B).

View larger version (18K): [in this window] [in a new window]   Fig. 8. A, effects of 10 µM THC on precontracted isolated resistance vessels (G3) over time compared with vehicle-treated control vessels. B, effects of the PPAR 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 S.E.M. *, P < 0.05; **, P < 0.01; ***, P < 0.001 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

View larger version (30K): [in this window] [in a new window]   Fig. 9. A, effects of THC (10 µM; 10 min) on the contractile response to methoxamine in isolated resistance arteries of the mesenteric bed (G3). B, effects of THC (10 µM; 2 h) on the contractile responses to methoxamine in the presence of catalase (2500 U/ml). C, effect of THC (10 µM; 2 h) on the contractile responses to methoxamine in the presence of 300 µM L-NAME. D, effects of THC (10 µM; 2 h) on the contractile responses to methoxamine in the presence of L-NAME and indomethacin in combination with apamin and charybdotoxin. E, effects of THC (10 mM; 2 h) on the contractile response to methoxamine in the presence of 10 µM indomethacin). F, effects of THC (10 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 S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

Incubation with 10 µM THC for 2 h inhibited the vasorelaxant responses to acetylcholine (vehicle pEC₅₀ = 7.96 ± 0.10 cf. THC pEC₅₀ = 7.49 ± 0.10; n = 5; P < 0.01; Fig. 1B). In the presence of L-NAME (Fig. 10A), THC greatly attenuated the vasorelaxant responses to acetylcholine. The effect of THC became more prominent with prolonged incubation, because although incubation for 10 min with THC caused some attenuation of the acetylcholine response, this was not significantly different from vehicle-treated vessels (vehicle pEC₅₀ = 7.83 ± 0.23 cf. THC pEC₅₀ = 7.58 ± 0.30; n = 7; Fig. 10B). In the presence of catalase, THC significantly inhibited the maximal vasorelaxant response to acetylcholine (vehicle R_max = 93.3 ± 3.6% relaxation cf. THC R_max = 73.8 ± 4.8% relaxation; n = 6; P < 0.01; Fig. 10C). 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 vessels (10 µM; 2 h) (vehicle R_max = 30.5 ± 2.2% relaxation cf. THC R_max = 34.4 ± 2.6% relaxation; n = 5; Fig. 10D). 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 = 10.4 ± 1.4% relaxation cf. THC R_max = 12.0 ± 1.0% relaxation; n = 3; Fig. 10D).

View larger version (21K): [in this window] [in a new window]   Fig. 10. A, representative trace showing the effects of THC compared with a vehicle-treated segment of the same artery on the vasorelaxant effects of acetylcholine in the presence of the NO synthase inhibitor L-NAME (300 µM). B, effects of THC (10 µM; 10 min) on the vasorelaxant response to acetylcholine. C, effects of THC (10 µM; 2 h) on the vasorelaxant response to acetylcholine in the presence of catalase (2500 U/ml). D, effects of THC (10 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 S.E.M. *, P < 0.05; **, P < 0.01 denotes significant difference between THC- and vehicle-treated adjacent segments of artery (paired Student's t test).

	Discussion

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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 seems to involve superoxide dismutase, the production of hydrogen peroxide, and calcium channel inhibition. Furthermore, in the superior mesenteric artery, incubation with THC for 2 h led to augmented endothelium-dependent vasorelaxation to acetylcholine, which was inhibited by PPAR 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 seemed 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., 2005a). Importantly, we showed that these effects were mediated by PPAR activation. These novel data have led to several questions as to whether THC has similar effects in the cardiovascular system as other PPAR ligands. Our present study first aimed to further characterize the in vitro effects of long-term THC exposure to establish whether preincubation with THC would lead to changes in agonist-stimulated responses as a consequence of altered protein activity caused by PPAR activation. We now show that in conduit arteries (the superior mesenteric artery and aorta), 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 because of changes in nitric oxide production. However, in the presence of catalase, which metabolizes H₂O₂ into water and oxygen and thus terminates the biological actions of H₂O₂, the effects of THC were inhibited, suggesting that THC inhibits contractile responses through increased H₂O₂ production. SOD catalyzes the conversion of superoxides to H₂O₂, 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 H₂O₂ 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 H₂O₂ production. In support of these data, it has been previously shown that incubation with H₂O₂ inhibits agonist-stimulated contractions, although the underlying mechanisms for this remained unclear (Iesaki et al., 1994). Additionally, a role for Ca²⁺ channel blockade has been implicated in the vasorelaxant effects of H₂O₂ 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 H₂O₂ might play a role in the modulation of calcium channels. To summarize, our data suggest that through an increase in SOD activity, THC stimulates H₂O₂ production, which leads to calcium channel blockade and subsequent inhibition of contractile responses.

In the superior mesenteric artery, it was found that incubation with THC for 2 h 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 because of increased stimulation of NO release, but it was inhibited in the presence of catalase and DETCA, again pointing toward a role for increased agoniststimulated H₂O₂ release through increased SOD activity. It has been previously shown that some of the endothelium-dependent vasorelaxant effects of acetylcholine are through the release of H₂O₂ in certain vessels (Matoba et al., 2000; Hatoum et al., 2005). Thus, the difference in results obtained between the aorta and superior mesenteric artery may be explained by the fact that in the aorta, H₂O₂ does not seem 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 H₂O₂ 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 H₂O_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 PPAR antagonist GW9662, which is in line with our previous finding that time-dependent vasorelaxation to THC is PPAR-mediated (O'Sullivan et al., 2005a). It has also previously been shown that PPAR ligand treatment augments/restores vasorelaxant responses to acetylcholine in various models of endothelial dysfunction, including diabetes (Kanie et al., 2003; Majithiya et al., 2005) and hypertension (Ryan et al., 2004). Additionally, a recent study has shown that PPAR ligands (ciglitazone or 15-deoxy-12,14-prostaglandin J2) stimulate both activity and expression of Cu/Zn-SOD in human umbilical vein endothelial cells (Hwang et al., 2005), which is also consistent with the present data. Because the time-dependent, SOD-dependent effects of THC on methoxamine-induced responses were PPAR-independent, it may be that THC is capable of increasing SOD activity and H₂O₂ production through both PPAR-dependent and -independent mechanisms. It is of note that 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, PPAR-mediated vasorelaxation of conduit arteries (O'Sullivan et al., 2005a). In the present study, we have examined whether a similar response to THC is observed in isolated resistance arteries of the mesenteric arterial bed. Although 10 µM THC caused the expected acute vasorelaxation of resistance vessels (O'Sullivan et al., 2005b), after 30 min, there was no difference between the THC-treated and vehicle-treated vessels; furthermore, PPAR antagonism had no effect on the THC response in resistance vessels at any time point. Thus, unlike in the aorta and superior mesenteric artery (O'Sullivan et al., 2005a), there is no time-dependent PPAR-mediated vasorelaxation to THC in resistance mesenteric arteries. Although our data do not point toward a reason for the heterogeneity between vessel types, it might be speculated that this could be because of differences in expression and/or function of the PPAR receptor between tissues. Because 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 10 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 shown previously in rabbit mesenteric arteries (Fleming et al., 1999) and mesenteric arteries (O'Sullivan et al., 2005b). 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 (Fig. 10B). However, when EDHF activity was blocked, there was no longer any difference between the vehicle- and THC-treated vessels. Together, this suggests that THC is capable of inhibiting both the basal and agonist-stimulated production of EDHF in resistance vessels.

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 predominant relaxing factor, THC has no effect on agonist-stimulated vasorelaxation. In the superior mesenteric artery, where hydrogen peroxide production contributes to vasorelaxation, THC enhances agonist-stimulated endothelium-dependent vasorelaxation. Finally, in resistance mesenteric arteries, where EDHF is the predominant relaxing factor, THC inhibits endothelium-dependent vasorelaxation.

In conclusion, we have now shown further time-dependent vascular action of THC in conduit arteries that are both PPAR-mediated and -independent, but both seem to involve increases in SOD activity, leading to increased H₂O₂ 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 thank Dr. Richard Roberts for the use of a myograph.

	Footnotes

 
This study was funded by the British Heart Foundation Grant PG2001/150.

doi:10.1124/jpet.105.095828.

ABBREVIATIONS: THC, ⁹-tetrahydrocannabinol; PPAR, peroxisome proliferator-activated receptor; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; L-NAME, N^G-nitro-L-arginine methyl ester; SOD, superoxide dismutase; DETCA, diethyldithiocarbamate; EDHF, endothelium-derived hyperpolarizing factor; ChTX, charybdotoxin; Veh, vehicle.

Address correspondence to: Dr. Saoirse E. O'Sullivan, School of Biomedical Sciences, E Floor, Queen's Medical Centre, University of Nottingham, NG7 2UH, UK. E-mail: saoirse.o'sullivan{at}nottingham.ac.uk

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