From the discovery of anandamide in the central nervous system and the identification of "classical" cannabinoid CE^ and CB2 receptors, both centrally and peripherally, the cardiovascular actions have received interest. To date there have been a number of studies which point to endocannabinoids having vasodilator actions, whereas the in vivo effects are less clear. One key point to emerge is that endocannabinoids may act via a range of mechanisms, of which action at vanilloid receptors is now well established.
The overwhelming theme from in vitro studies is that endogenous cannabinoids cause vasorelaxation. However, as will be seen later, this does not simply translate into the in vivo situation. The first in vitro report that anandamide was a vasodilator came from Ellis et al. (1995), who demonstrated that anandamide caused cerebrovascular vasodilatation. Subsequent studies also demonstrated that anandamide was a vasorelaxant in the rat isolated mesenteric and coronary vasculatures (Randall et al., 1996; Randall and Kendall, 1997).
In the Ellis et al. study, anandamide was shown to act via the release of vasodilator prostanoids, and A9-tetrahydrocannabinol (A9-THC) also acted in this way. Furthermore, Fleming et al. (1999) found that the cyclooxygenase inhibitor, diclofenac, abolished vasorelaxation to anandamide in rat
mesenteric arterial vessels. In addition, Grainger and Boachie-Ansah (2001) reported that, in the sheep coronary artery, anandamide caused relaxations and this involved cyclooxygenase-dependent metabolism to vasodilator prostanoids. Despite these observations, most other studies have ruled out a major role for prostanoids in anandamide-induced relaxation (Randall et al., 1996; Randall et al., 1997; Plane et al., 1997; White and Hiley, 1997).
The role of the endothelium in vasorelaxation to endogenous cannabinoids varies between vascular beds and tissues. Most studies have shown that the vasorelaxant responses to anandamide are endothelium-independent (Randall et al., 1996; White and Hiley, 1997; White et al., 2001) or only partly endothelium-dependent (Chaytor et al., 1999). However, in the bovine coronary artery, anandamide induces relaxations that are strictly endothelium-dependent (Pratt et al., 1998). This was explained by the endothelial cells metabolizing exogenous anandamide, via a cytochrome P450-dependent mono-oxygenase, to vasoactive metabolites. Similarly, Grainger and Boachie-Ansah (2001) reported endothelium-dependent metabolism of anandamide via the cyclooxygenase pathway underpinning vasorelaxation.
In 1999, Wagner and colleagues proposed that anandamide acted, in part, via an endothelial anandamide receptor in rat mesenteric arterial vessels. This was based on the observation that relaxation to anandamide was partly sensitive to both removal of the endothelium and the CBj receptor antagonist SR141716A, but when the endothelium was removed, the sensitivity to the antagonist was lost. This led to the proposal that anandamide acted at a cannabinoid receptor that was sensitive to SR141716A but as it was not the CBj receptor, it was termed the "anandamide receptor." An additional observation was that the exogenous cannabinoid A9-THC did not cause vasorelaxation. Subsequent work by that group demonstrated that the endothelial cannabinoid receptor was also activated by the neurobehaviorally inactive "abnormal cannabidiol" (abn-cbd), which caused vasorelaxation (Jarai et al., 1999). One possibility to arise from the identification of the SR141716A-sensitive, endothelium-dependent component is that anandamide acts in part via EDHF and that SR141716A is acting via inhibition of EDHF activity (e.g., through blockade of myoendothelial gap junctions, Chaytor et al., 1999).
In 2003, Offertaler et al. provided further evidence for the "endothelial anandamide" receptor. Specifically, they reported that a novel cannabidiol analog, 0-1918, opposed the relaxant effects of anandamide and abn-cbd, the in vivo hypotensive effects of abn-cbd, and the phosphorylation of p42/44 MAP kinase induced by abn-cbd in endothelial cells. These actions of 0-1918 were independent of classical cannabinoid and vanilloid receptors, and this led the authors to conclude that 0-1918 was a selective antagonist of the "endothelial anandamide" receptor. It was suggested that the endothelium-dependent relaxation to abn-cbd and anandamide is G protein coupled to MAP kinase activation and charybdotoxin-sensitive potassium channels but not to nitric oxide. Taken together, the authors proposed that the novel receptor may be coupled to the release of the EDHF.
The identification of EDHF is controversial, with several agents being proposed as representing EDHF. These include cytochrome P450-derived metabolites of arachidonic acid, potassium ions, and mediation via myoendothelial gap junctions (Busse et al., 2002). In relation to myoendothelial gap junctions, Chaytor et al. (1999) demonstrated that the actions of anandamide in rabbit mesenteric vessels were partly endothelium-dependent and sensitive to gap junctional inhibitors, and that SR141716A is a gap junctional inhibitor. These findings certainly point to anandamide acting in part via EDHF-type relaxation, which involves myoendothelial gap junctions. Furthermore, in the rat mesenteric arterial bed, Harris et al. (2002) reported that some but not all gap junction inhibitors opposed responses to anandamide, although an alternative possibility was that these inhibitors were inhibiting the sodium pump.
It was originally proposed that anandamide itself was an EDHF (Randall et al., 1996). This was partly based on the observation that SR141716A opposed EDHF-type relaxations. This inhibitory action was explained by the work of Chaytor et al. (1999), which demonstrated that the cannabinoid CBj receptor antagonist SR141716A was also a gap junctional inhibitor and was thus blocking EDHF activity at this level, and that EDHF-type relaxations are involved in the responses to anandamide. Similarly, sensitivity of relaxant responses to potassium channel blockers, including cytochrome P450 inhibitors (Randall et al., 1997) charybdotoxin and iberiotoxin (Plane et al., 1997), and the combination of charybdotoxin and apamin (Randall and Kendall, 1998) can be explained by EDHF's mediating part of the relaxation to anandamide. However, 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, glibenclamide (Chataigneau et al., 1998), which may implicate these channels in the responses to anandamide, an action independent of EDHF.
Anandamide has been shown to act via the release of endothelium-derived nitric oxide in the rat kidney (Deutsch et al., 1997). A range of human blood vessels and the right atrium have also been shown to release nitric oxide in response to anandamide (Bilfinger et al., 1998). However, in many instances (see Randall et al., 1996; White and Hiley, 1997; Jarai et al., 1999), vasorelaxant responses to anandamide are insensitive to inhibition of nitric oxide synthase. In HUVECs, Maccarrone et al. (2000) reported that anandamide and the CB agonist, HU210, both cause an upregulation of the expression and activity of the inducible nitric oxide synthase. Mukhopadhyay et al. (2002) demonstrated that the endothelium-dependent component was G protein coupled and mediated via nitric oxide, whereas the endothelium-independent component was due to activation of vanilloid receptors, at least in rabbit aortic rings.
Endocannabinoids have been shown to inhibit vascular, smooth-muscle calcium channels (Gebreme-dhin et al., 1999). Specifically, in feline cerebral vessels, it was shown that endocannabinoids and synthetic cannabinoid agonists act via G-protein-coupled CB1 receptors to cause inhibition of voltage-sensitive calcium channels, leading to vasodilatation. This action was proposed to contribute towards vasodilatation in cerebral hypoxia, which was associated with the release of endocannabinoids.
One of the most attractive and novel proposals to account for the vasodilatation in response to anand-amide has been that it acts as a vanilloid agonist, because anandamide shares structural similarities with the vanilloid agonist olvanil. This led Zygmunt et al. (1999) to investigate the role of vanilloid receptors in the vascular actions of anandamide. In this respect, they reported that relaxation to anandamide (but not 2-AG, palmitoylethanolamide, or synthetic cannabinoid receptor agonists) was essentially abolished by depletion of the sensory nerves of calcitonin gene-related peptide (CGRP) by capsaicin in guinea pig basilar, rat hepatic, and rat mesenteric arteries. Furthermore, relaxation to anandamide was sensitive to the vanilloid receptor antagonist capsazepine and also to CGRP receptor antagonism with CGRP (8-37). Clearly, anandamide can evoke the release of neurotransmitters from sensory nerves leading to vasorelaxation. This conclusion was supported by the demonstration that anandamide is an agonist at the cloned rat vanilloid receptor (rVR1) (Zygmunt et al., 1999). This observation was later confirmed at the human vanilloid receptor (hVR1) (Smart et al., 2000).
Similar observations have been made with the analog of anandamide, methanandamide, which was also shown to cause capsaicin- and capsazepine-sensitive vasorelaxation in the rat mesenteric arterial bed and isolated mesenteric arteries (Ralevic et al., 2000). However, in the same vascular bed, Harris et al. (2002) reported that vasorelaxation to anandamide was only partly sensitive to capsaicin pretreatment. Moreover, in the presence of NO synthase blockade, vasorelaxation due to anandamide was insensitive to capsaicin pretreatment and thus does not occur exclusively via sensory nerves. In rat isolated coronary arteries and in the intact vasculature, White et al. (2001)
and Ford et al. (2002) have reported that vasorelaxation to anandamide is independent of vanilloid VR1 receptors and appears to be mediated via a novel cannabinoid receptor. Accordingly, the activation of sensory nerves by anandamide may only explain part of the actions of anandamide and only under some circumstances.
More recent work by Zygmunt et al. (2002) has shown in rat mesenteric vessels that A9-THC and cannabinol cause relaxation via action on sensory nerves but that this involves a site of action which is not VR1. From this, they propose that there is a novel cannabinoid receptor or target which may control sensory nerve transmission.
The fact that the hypotensive action of anandamide is absent in knockout mice lacking CB1 receptors (Ledent et al., 1999) suggests that any action via vanilloid receptors on sensory nerves is only of minor importance in the hemodynamic profile of systemically administered cannabinoids. In urethane-anesthetized rats, there is a triphasic cardiovascular response to systemic intravenous administration of anandamide, but it appears that only the initial rapid hypotensive and bradycardic phase involves VRj receptors (Malinowska et al., 2001, Smith and McQueen, 2001), and this phase is not associated with vasodilatation (Gardiner et al., 2002a).
^ Unifying Mechanism of Vasorelaxation?
The preceding sections have dealt with a range of possible mechanisms of vasorelaxation to endogenous cannabinoids. There is clearly a diverse range of putative mechanisms, and this may well reflect tissue differences. Because it is known that EDHF activity tends to be greatest in smaller resistance vessels, it might be speculated that EDHF might play a significant role in these vessels, and the work of Offertaler et al. (2003) supports the notion that the release of EDHF is coupled to a novel endothelial cannabinoid receptor. Undoubtedly, VRj receptors on sensory nerves also play a substantial role in mediating responses to endogenous cannabinoids; once again, contribution may differ between different arteries and, indeed, prevailing conditions. In relation to tissue selectivity, Andersson and colleagues (2002) have addressed this issue and, in this regard, suggest that differences in the abundance of the cannabinoid transporter, and the relative content of cannabinoid and vanilloid receptors, coupled with agonist efficacy, may determine which mechanisms prevail.
Contractions mediated by anandamide and synthetic cannabinoids have been reported in rat isolated small mesenteric arteries (White and Hiley, 1998). The responses were small, and it was suggested that only low levels of calcium were released from intracellular stores, which were insufficient to stimulate extracellular calcium entry. In that rat aorta, A9-THC has been shown to cause contractile responses that are mediated by cyclooxygenase metabolites (O'Sullivan et al., 2005). In the anesthetized rat, anandamide, despite causing widespread vasodilatation, has recently been shown to cause SR141716A-insensitive vasoconstriction in the spleen (Wagner et al., 2001b). It is possible that synergistic interactions of cannabinoids with circulating and locally released contractile mediators account for the pronounced, albeit transient, pressor response observed upon systemic administration of cannabinoids in anesthetized animals.
The ability of cannabinoids to cause vascular effects implies that the vasculature contains a molecular target. Evidence to date suggests that there may be vascular cannabinoid receptors, which may either fall into the classical CB1/CB2 classification or represent a new subtype. As stated above, the biphasic hypotension in response to anandamide is absent in CB1 receptor knockout mice (Ledent et al., 1999). This clearly points to the involvement of the CB1 receptor. However, it should be noted that this does not conclusively identify the cannabinoid receptors as being on the vascular smooth muscle or associated with neuronal tissue. The sensitivity of vasorelaxant responses to CB receptor antagonists has been controversial, with some studies indicating that the responses are opposed by SR141716A (Randall et al., 1996; White and Hiley, 1997) and others demonstrating that they are insensitive to this antagonist (Plane et al., 1997). The insensitivity to SR141716A might reflect noncannabinoid receptor actions (Pratt et al., 1998; Chaytor et al., 1999), whereas Jarai et al. (1999) have suggested the presence of a novel vascular CB receptor.
Using reverse transcriptase-polymerase chain reaction, the gene product encoding for CB! receptors has been located in renal endothelial cells, mesenteric resistance arterioles, and cerebral micro vessels, which is consistent with the expression of CB! receptors in the vasculature (Deutsch et al., 1997; Darker et al., 1998; Randall et al., 1999). Others have also identified mRNA in human endothelial cells (Sugiura et al., 1998; Liu et al., 2000) and Liu et al. (2000) have identified CB receptor binding sites by radioligand studies. Similarly, immunoreactivity to the CBi receptor has been identified on human saphenous vein endothelial cells (Bilfinger et al., 1998).
Cannabinoid CBi receptors have also been localized to cat cerebral arterial smooth muscle (Gebremedhin et al., 1999). In this study, it was demonstrated that feline vascular smooth muscle contained CBi receptors together with cDNA, showing very close homology to that associated with neuronal CBi receptors.
Not only do endocannabinoids affect vascular function, but they may also have direct cardiac actions. Ford et al. (2002) reported in the rat isolated heart that anandamide had a negative inotropic effect and reduced left ventricular pressure. This action appeared to be due to action at a novel cannabinoid receptor. In human atrial muscle, anandamide has also been shown to exert negative inotropic effects but, in this case, via the activation of CBi receptors (Bonz et al., 2003).
In vivo, the cardiovascular effects of exogenous cannabinoids are variable, with both vasodilator and vasoconstrictor actions being reported (Stark and Dews, 1980). In humans, acute administration of cannabinoids is associated with tachycardia and a small pressor effect; whereas long-term use is associated with hypotension and bradycardia (Benowitz and Jones, 1975; Benowitz et al., 1979). In 1996, Vidrio and colleagues reported that the cannabinoid agonist HU210, following intraperitoneal administration, caused prolonged bradycardia and hypotension, in both conscious and anesthetized rats. The hypotension in response to HU210 in anesthetized rats was subsequently reported by Wagner et al. (2001b) as being due to a reduction in cardiac output without effects on vascular resistance. Work in pithed rabbits by Niederhoffer and Szabo (1999), in which sympathetic tone was evoked by continuous electrical stimulation, has demonstrated that intravenous injection of CBi receptor agonists (CP55940 and WIN55212-2) causes prejunctional inhibition of sympathetic activity leading to hypotension. By contrast, Gardiner et al. (2002b) demonstrated in conscious rats that WIN55212-2 and HU210 caused pressor and regional vasoconstrictor effects. These effects were sensitive to the cannabinoid CBi receptor antagonist AM251 and appeared to be mediated via increased sympathetic activity. In addition, the cannabinoid agonists also caused hindquarters vasodilatation via the activation of (^-adrenoceptors. These clear differences with previous findings were ascribed to the confounding effects of generic anesthetic agents used in most in vivo studies.
The cardiovascular effects of endogenous cannabinoids are similarly complex; with respect to anandamide in anesthetized rats, it has been shown to cause bradycardia (with brief secondary hypotension), then a transient pressor effect, which is followed by a delayed but long-lasting depressor action (Varga et al., 1995; Lake et al., 1997). The initial bradycardia and associated hypotension are believed to be vagally mediated, as it is abolished by atropine treatment or cervical vagotomy (Varga et al., 1995).
In the anesthetized rat, the second depressor effect, which follows the transient pressor phase, is believed to be mediated by CBi receptor prejunctional inhibition of sympathetic outflow in the periphery as the effect is attenuated by cervical spinal transection, a-adrenoceptor, and cannabinoid receptor antagonists (Varga et al., 1995; Lake et al., 1997).
In conscious rats, the cardiovascular effects of anandamide are markedly different from those reported in studies carried out in anesthetized animals. In conscious rats, Stein et al. (1996) reported that anandamide caused bradycardia, with a transient hypotensive effect, followed by a longer pressor phase, and only at the higher doses was there delayed hypotension. It seems likely that the greater pressor effect obscures the hypotension. Furthermore, Gardiner and colleagues (2002a) reported that intravenously administered anandamide led to transient pressor effects associated with mesenteric, renal, and hindquarters vasoconstriction. High doses of anandamide were associated with initial bradycardia, and the hindquarters vasoconstriction was followed by vasodilatation. These complex cardiovascular actions were insensitive to the CBj receptor antagonist, AM251 and thus, not mediated via cannabinoid CB1 receptors. The bradycardia was atropine-sensitive, and the apparent hindquarters vasodilatation appeared to be mediated via ^-adrenoceptors. It was speculated that this may be due to the release of adrenaline via adrenal vanilloid receptors.
In mice, both anandamide and synthetic cannabinoid receptor agonists cause biphasic hypotension (a depressor response, followed by a more sustained hypotensive phase but without a pressor component), which is thought to be entirely CB1 receptor mediated, as the responses are absent in CB1 receptor knockout mice (Ledent et al., 1999).
Wagner et al. (1997) demonstrated in a rat model of hemorrhagic shock that activated macrophages release anandamide. In endotoxic shock, the synthesis of 2-AG in platelets is increased and anandamide is only detectable in macrophages after exposure to lipopolysaccharide (Varga et al., 1998). In vitro, mouse J774 macrophages also release both 2-AG and anandamide, and participate in their degradation (Di Marzo et al., 1999). In patients with endotoxic shock, increases in plasma anandamide and 2-AG have now been reported (Wang et al., 2001). These findings certainly point to the genesis of endocannabinoids in blood cells, which is enhanced in shock and contributes towards the cardiovascular sequelae.
In the context of septic shock, the induction of the inducible nitric oxide synthase and excessive production of nitric oxide are widely implicated. Interestingly, Ross et al. (2000) demonstrated that the cannabinoid agonist WIN55212, acting via CB2 receptors, actually inhibited lipopolysaccharide-induced nitric oxide release from macrophages.
In terms of hemorrhagic shock, Wagner et al. (1997) demonstrated that the accompanying hypotension, in part due to macrophage-derived endocannabinoids, was reversed by the cannabinoid receptor antagonist SR141716A. Similarly in endotoxic shock, the synthesis of 2-AG in platelets and anandamide in macrophages is increased (Varga et al., 1998). It is possible that the activated blood cells could also stimulate the release of endocannabinoids from the endothelium or other vascular sites, contributing further towards the hypotension. The release of anandamide by central neurones under hypoxic conditions, leading to improved blood flow and protection against ischemia, has also been advanced as a pathophysiological role for anandamide (Gebremedhin et al., 1999).
There is also the possibility that pathophysiological conditions may alter the normal responses to endocannabinoids. In this regard, Mendizabal and colleagues (2001) reported that the vasorelaxant responses to anandamide were enhanced in mesenteric vessels from rats rendered hypertensive through chronic treatment with a nitric oxide synthase inhibitor but not in rats rendered hypertensive through aortic coarctation. Enhanced responses following chronic nitric oxide synthase inhibition were confirmed by Tep-areenan et al. (2002). Others have also reported that depressor responses to anandamide are enhanced in anesthetized, spontaneously hypertensive rats (SHR) (Li et al., 2003). It is thus possible that the endocannabinoid system may become altered to compensate for this loss of nitric oxide.
Endotoxemia is another circumstance in which responses to anandamide may be altered, as Orliac et al. (2003) reported that vasorelaxation to anandamide was enhanced in mesenteric arterial beds from endotoxemic rats. They concluded that this upregulation might involve a metabolite of anandamide and also activation of vanilloid receptors.
As commented upon in the preceding text, endocannabinoids may exert cardiac effects, and there has been interest in the influence of cannabinoids on cardiac ischemia. Lagneux and Lamontagne (2001) reported that cardioprotection of the rat heart against ischemia by pretreatment with lipopolysaccharide involved endocannabinoids. In this respect, pretreatment with lipopolysaccha-ride was found to protect hearts against ischemia, but this was sensitive to blockade via the cannabinoid CB2 receptor antagonist SR144528 but not the CBj receptor antagonist SR141716A. The implication from this work was that lipopolysaccharide-induced cardioprotection involved the release of endocannabinoids, which caused protection via cannabinoid CB2 receptors. Subsequent work by that group also reported that palmitoylethanolamide and 2-arachidonoyl glycerol both caused cardioprotection via CB2 receptor activation and that this involved p38, ERK1/2, and protein kinase C activation (Lepicier et al., 2003).
Related to cardioprotection, Wagner and colleagues (2001a) have reported that in a rat model of myocardial infarction due to coronary ligation in vivo, there is the release of anandamide and 2-arachidonoyl glycerol from monocytes and platelets. This release of endocannabinoids was associated with systemic hypotension but decreased mortality, as administration of SR141716A reduced the hypotension but increased mortality. This might suggest that the release of endocan-nabinoids may be protective. In this model, the release of endocannabinoids did not reduce the size of myocardial infarction. Subsequent studies have examined the effects of treatment of the CBj receptor antagonist AM251 or the CB agonist HU210 for 12 weeks after myocardial infarction (Wagner et al., 2003). The key findings from this study were that cannabinoid receptor antagonism promotes remodeling and that cannabinoid agonists may prevent endothelial dysfunction and hypotension. Therefore, it is possible that the release of endocannabinoid in postmyocardial infarction might play a role in opposing the deleterious process of remodeling, which leads to long-term complication such as the development of heart failure. It was also observed that the administration of the cannabinoid agonist led to preservation of endothelial function.
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