Mechanisms of vasorelaxation for endocannabinoids

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The original findings suggested that anandamide might act via a hyperpolarizing mechanism (Randall et al., 1996). This proposal was confirmed electrophysiologi-cally by the demonstration that anandamide causes hyperpolarization or repolar-ization of vascular smooth muscle, but in both cases this effect was independent of cannabinoid CBX receptors (Plane et al., 1997, Chataigneau et al., 1998). The hyperpolarization was also found to be endothelium-dependent (Chataigneau et al., 1998; Zygmunt et al., 1997), with the implication that anandamide acted via the release of EDHF (Figure 20.3). The latter study also provided evidence that anandamide acted via inhibition of calcium mobilization in vascular smooth muscle cells, without direct effects on potassium conductance.

In rat mesenteric vessels, anandamide-induced relaxation is sensitive to nonspecific potassium channel blockers, including cytochrome P450 inhibitors (Randall et al., 1997). In isolated mesenteric arterial segments the relaxation to anandamide was blocked by selective inhibitors of large conductance calcium-activated K+-channels (charybdotoxin and iberiotoxin; Plane et al., 1997). Furthermore, in similar mesenteric vessels, the anandamide-induced relaxation was insensitive to

Figure 20.3 Summary diagram of the vascular pharmacology of endocannabinoids, incorporating the various putative sites of synthesis, and proposed mechanisms and sites of action. (1) The proposal that anandamide (ana) acts on novel endothelial cannabinoid receptors (CBx) to elicit the release of the endothelium-derived hyperpolarizing factor (EDHF) (Wagner et al., 1999; Jarai etal., 1999). (2) The suggestion that anandamide gains access to the endothelium via the transporter and acts via gap junctions (Chaytor et al., 1999). (3) The proposal that the endothelium releases endocannabinoids (Randall et al., 1996; Deutsch et al., 1997; Sugiura et al., 1998). The actions of anandamide via: (4) activation of the potassium channels (Randall etal., 1996; Plane et al., 1997; Randall et al., 1997; Randall and Kendall, 1998; Chataigneau et al., 1998; Ishioka and Bukoski, 1999); (5) inhibition of voltage-operated calcium channels (Gebremedhin etal., 1999) (both 4 and 5 via coupling (6) to CB-recep-tors); (7) activation of the sodium pump (Figure 20.4). (8) Illustrates the release of anandamide from macrophages in shock (Wagner et al., 1997; Varga et al., 1998). (9) The proposal that anandamide acts via vanilloid receptors (VR) to release CGRP from sensory nerves (Zygmunt et al., 1999). (10) The proposal that sensory nerves release anandamide (Ishioka and Bukoski, 1999).

the combination of charybdotoxin and apamin (White and Hiley, 1997). By contrast in the perfused mesenteric arterial bed the combination of charybdotoxin and apamin abolished relaxation to anandamide, although neither agent alone affected the responses (Randall and Kendall, 1998). In the guinea-pig carotid artery, the anandamide-induced hyperpolarization, which was insensitive to charybdotoxin plus apamin, was blocked by the ATP-sensitive potassium channel inhibitor, glib-enclamide (Chataigneau et al., 1998). By contrast, glibenclamide does not affect anandamide-induced relaxation in the rat mesentery (Randall et al., 1997; White and Hiley, 1997). In conclusion some, but not all, studies point to the involvement of K+-channels, at some stage, in the vasorelaxant actions of anandamide (Figure 20.3).

The endothelium-dependent hyperpolarization and sensitivity, in some cases, to K+-channel inhibitors, raised the possibility that anandamide might act in an endothelium-dependent manner via the release of EDHF. However, in this respect initial studies indicated that vasorelaxation was preserved following removal of the endothelium (Randall et al., 1996; White and Hiley, 1997; Figure 20.2). By contrast this was not found to be the case in all blood vessels. In bovine coronary vessels anandamide certainly causes endothelium-dependent relaxation by metabolism to cytochrome P450 metabolites of arachidonic acid (Pratt et al., 1998). In rabbit mesenteric vessels anandamide acts partly in an endothelium-dependent manner following uptake into the endothelial cells, where it appears to promote gap junctional opening, leading to smooth muscle relaxation (Chaytor et al., 1999). Endo-thelial uptake may also be important in allowing anandamide to have intracellular effects, such as raising cytosolic calcium (Mombouli et al., 1999). This action itself might trigger endothelial-mediated responses, such as the release of EDHF.

In rat mesenteric vessels, Wagner et al. (1999) identified a small endothelial component of relaxation to anandamide which was SR 141716A-sensitive but not mediated by CBj-receptors. This led to them to propose that there is a novel endothelial cannabinoid receptor (Figure 20.3). One alternative explanation for this could be that SR141716A was acting to inhibit responses to anandamide via inhibition of gap junctions (Chaytor et al., 1999). Further work in this area has indicated that a neurobehaviorally inactive cannabinoid, abnormal cannabidiol, causes SR141716A-senstive mesenteric vasodilatation which is also blocked by can-nabidiol (Jarai et al., 1999). From these findings it was proposed that cannabidiol was an antagonist of this novel endothelial cannabinoid receptor, which is coupled to EDHF release (Figure 20.3).

Under some circumstances anandamide has been shown to act via release of endothelium-derived nitric oxide (Deutsch et al., 1997), although in many instances (see Randall et al., 1996; White and Hiley, 1997; Jarai et al., 1999) the responses to anandamide are insensitive to inhibition of nitric oxide synthase.

The possibility that vasorelaxation to anandamide might involve EDHF release led us to investigate further the pharmacology of vasorelaxation to anandamide. To this end the effects of gap junction inhibitors and ouabain, the sodium pump inhibitor which also inhibits gap junctional communication, were investigated against responses to anandamide (Figure 20.4). In this respect we were unable to demonstrate any endothelial-dependence of vasorelaxant responses to ananda-mide. However, the vasorelaxation was sensitive to gap junction inhibitors (18a-glycyrrhetinic acid and ouabain) which also block the sodium pump but was unaffected by agents which are selective for gap junctions (carbenoxolone and palmitoleic acid). This has raised the possibility that the sodium pump may at some stage be involved in vasorelaxation to anandamide independently of any contribution of EDHF.

Vascular smooth muscle calcium channels have also been proposed to be the target for endocannabinoids (Gebremedhin et al., 1999; Figure 20.3). In feline cerebral vessels it was shown that both endocannabinoids and synthetic

Figure 20.4 Vasorelaxation to anandamide in the rat isolated perfused mesenteric arterial bed in the presence of the combined gap junction and sodium pump inhibitors (1mM ouabain and 100 |J,M 18 a-glycyrrhetinic acid (18a-GA)) and pure gap junction inhibitors (50 |aM palmitoleic acid (PA) and 100 |aM carbenoxolone). These data support the proposal that anandamide may act via activation of the sodium pump but not via gap junction activation. Data shown as mean ± S.E.M.

Figure 20.4 Vasorelaxation to anandamide in the rat isolated perfused mesenteric arterial bed in the presence of the combined gap junction and sodium pump inhibitors (1mM ouabain and 100 |J,M 18 a-glycyrrhetinic acid (18a-GA)) and pure gap junction inhibitors (50 |aM palmitoleic acid (PA) and 100 |aM carbenoxolone). These data support the proposal that anandamide may act via activation of the sodium pump but not via gap junction activation. Data shown as mean ± S.E.M.

cannabinoids act via G-protein coupled CBrreceptors to cause inhibition of voltage-sensitive calcium channels, leading to vasodilatation.

One of the most recent proposals for the vascular actions of anandamide has been that anandamide is a vanilloid agonist. In support of this, Zygmunt et al. (1999) reported that vasorelaxant responses to anandamide (but not 2-AG, palmi-tylethanolamide or synthetic cannabinoid receptor agonists) were essentially abolished by pre-treatment with capsaicin to deplete the sensory nerves, especially of calcitonin gene-related peptide (CGRP) (Figure 20.3). In addition, the responses to anandamide were sensitive to the vanilloid antagonist capsazepine and, also, CGRP receptor antagonism. The conclusion from this study was that anandamide evoked the release of transmitters from sensory nerves leading to vasorelaxation. In support of this, similar observations have been made with the analog of anandamide, methanandamide (Ralevic et al., 2000). By contrast, Harris et al. (2000) have reported that the responses to anandamide were only partly sensitive to capsaicin pre-treatment in the presence of a functional NO system. In the presence of NO synthase blockade vasorelaxation due to anandamide was insensitive to capsaicin pretreatment and thus does not occur via sensory nerves. Accordingly the activation of sensory nerves by anandamide may only explain part of the actions anandamide, and only under some circumstances. Indeed, the fact that the hypotensive action of anandamide is absent in mice lacking CB1 receptors (Ledent et al., 1999) certainly suggests that any action via vanilloid receptors is only of minor importance.

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