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Further Characterization Of The Time-Dependent Vascular Effects Of Δ9-THC

Truth Seeker

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Abstract

We have previously shown that over time (2 h), the active ingredient of cannabis, Δ9-tetrahydrocannabinol (THC), produces peroxisome proliferator-activated receptor (PPAR) γ-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 PPARγ-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 PPARγ antagonist, 2-chloro-5-nitro-N-phenylbenzamide (GW9662), catalase, and DETCA, but not by the NO synthase inhibitor NG-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.

In the early 1990s, a receptor for the active constituent of marijuana, Δ9-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., 2004). 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., 2005b), the endothelium (Fleming et al., 1999; O'Sullivan et al., 2005b), sensory nerves (Zygmunt et al., 2002), and inhibition of calcium channels in combination with activation of potassium channels (O'Sullivan et al., 2005b).

Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear receptors of which there are three isoforms: α, δ, and γ (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). 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, 2000). PPARγ 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 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., 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 PPARα (Fu et al., 2003), and metabolism of another endocannabinoid, 2-arachidonoylglycerol, causes release of 15-hydroxyeicosatetraenoic acid glyceryl ester, a PPARα agonist (Kozak et al., 2002). Similarly, the endocannabinoid palmitolyethanolamide has anti-inflammatory properties mediated by PPARα (Lo Verme et al., 2005). It has also been reported that ajulemic acid, a THC metabolite analog, binds to PPARγ with potential anti-inflammatory actions (Liu et al., 2003). 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., 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

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 NG-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 PPARγ activation was investigated using the PPARγ 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 (Rmax) 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). Rmax 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.

Drugs. All drugs were supplied by Sigma Chemical Co. (Poole, Dorset, UK) except where stated. GW9662 was obtained from Tocris Cookson Inc. (Bristol, 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 100 μl of ethanol and then dissolved into the Krebs-Henseleit solution. THC was dissolved in ethanol at 10 mM with further dilutions made in distilled water. GW9662 was dissolved in dimethyl sulfoxide to 10 mM, with further dilutions made in distilled water.

Results

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 (Tables1and2). 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 Rmax = 1.74 ± 0.18 g cf. THC 2-h and washout Rmax = 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 PPARγ 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).

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).

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 PPARγ 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 (Tables1and2). 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 PPARγ antagonist GW9662 (n = 6; Fig. 5D).

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).

After 2-h incubation with 10 μM THC, the maximal vasorelaxant response (Veh Rmax = 99.2 ± 7.4% relaxation, THC Rmax = 130 ± 12% relaxation; n = 8; P < 0.05; Fig. 7A) to acetylcholine was significantly enhanced compared with vehicle-treated vessels, but this was not seen after short-term incubation (10 min) with THC (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 Rmax = 14.6 ± 1.5% relaxation, THC Rmax = 35.1 ± 2.5% relaxation; n = 6; P < 0.01; 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 Rmax = 8.4 ± 1.5% 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 10 μM THC 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. Two-hour 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, 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 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, because a similar enhancement of responses was seen after 10-min incubation with THC (vehicle pEC50 = 5.16 ± 0.15 cf. THC pEC50 = 5.60 ± 0.06; n = 7; P < 0.05; Fig. 9A). Similarly, in the presence of 10 μM indomethacin, THC enhanced the contractile responses to methoxamine (vehicle pEC50 = 5.75 ± 0.06 cf. THC pEC50 = 6.11 ± 0.14; n = 7; P < 0.05; Fig. 9E). In the presence of 300 μM L-NAME, 10 μ THC for 2 h had no effect on the contractile response to methoxamine (vehicle pEC50 = 6.22 ± 0.20 cf. THC pEC50 = 6.37 ± 0.21; n = 7; Fig. 9C). In the presence of catalase, there was no difference in the pEC50 of methoxamine between the vehicle-treated and THC-treated vessels (vehicle pEC50 = 5.98 ± 0.08 cf. THC pEC50 = 6.27 ± 0.17; 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 (300 nM methoxamine; Veh 0.08 ± 0.06 g increase in tension cf. THC 0.65 ± 0.22 g increase in tension; n = 7; P < 0.05; Fig. 9B). Following EDHF inhibition (L-NAME, indomethacin, ChTX, and apamin), THC no longer enhanced the contractile effects of methoxamine (vehicle pEC50 = 5.95 ± 0.20 cf. THC pEC50 = 5.93 ± 0.15; n = 6; Fig. 9D). In the presence of ChTX and apamin alone, there was no difference in the potency of methoxamine between vehicle- and THC-treated vessels (vehicle pEC50 = 6.05 ± 0.09 cf. THC pEC50 = 6.10 ± 0.24; n = 6; Fig. 9F). However, THC did cause a reduction in the maximal response to methoxamine (vehicle Rmax = 2.32 ± 0.14 cf. THC Rmax = 1.54 ± 0.23; n = 6; P < 0.05; Fig. 9F).

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 Rmax = 93.3 ± 3.6% relaxation cf. THC Rmax = 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 Rmax = 30.5 ± 2.2% relaxation cf. THC Rmax = 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 Rmax = 10.4 ± 1.4% relaxation cf. THC Rmax = 12.0 ± 1.0% relaxation; n = 3; Fig. 10D).

In summary, incubation with 10 μM THC 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.

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 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 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 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 PPARγ activation. These novel data have led to several questions as to whether THC has similar effects in the cardiovascular system as other PPARγ 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 PPARγ 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 H2O2.

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 (O'Sullivan et al., 2005a). 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., 2003; Majithiya et al., 2005) and hypertension (Ryan et al., 2004). Additionally, a recent study has shown that PPARγ ligands (ciglitazone or 15-deoxy-Δ12,14-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 PPARγ-independent, it may be that THC is capable of increasing SOD activity and H2O2 production through both PPARγ-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, PPARγ-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, PPARγ 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 PPARγ-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 PPARγ 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 PPARγ-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.

Source, Graphs and Figures: Further Characterization of the Time-Dependent Vascular Effects of Δ9-Tetrahydrocannabinol
 
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