AEA and Vanilloid VR1 Receptors

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A six-trans-membrane domain, nonselective cation channel, the vanilloid receptor of type 1 (VR1 or TRPV1) (Caterina et al., 1997) is the first and only member of the family of the TRP channels discovered so far to be activated by capsaicin, the pungent component of "hot" chili peppers (Holzer,

1991), as well as by another plant toxin, resiniferatoxin (RTX) (Szallasi and Blumberg, 1999) (Figure 6.2). VR1 acts as a ligand-, proton-, and heat-activated molecular integrator of nociceptive stimuli (Tominaga et al., 1998) and, as such, is mostly expressed in sensory C (and, to a lesser extent, A-6) fibers (Guo, 1999). Although originally identified as a plasma membrane protein, VR1 was recently shown to be also present on the endoplasmic reticulum, where it might participate in intracellular calcium mobilization (Liu et al., 2003). VR1 receptor knockout mice (Caterina et al., 2000; Davis, 2000) exhibited lower sensitivity to inflammatory and thermal pain, thus demonstrating the involvement of this protein in the transduction of inflammatory and thermal hyperalgesia. However, activation of VR1 by capsaicin and, particularly, RTX is almost immediately followed by desensitization. For this reason, some synthetic long-chain capsaicin analogs do not exhibit pungent activity and have been proposed as oral anti-inflammatory and analgesic compounds (Dray,

1992). VR1 is also expressed in several brain areas of primates and rodents, including the hippocampus, striatum, hypothalamus, substantia nigra compacta, and locus coeruleus (Cortright et al., 2001; Hayes et al., 2000; Mezey et al., 2000). This finding suggested the existence of endogenous ligands for vanilloid receptors (Kwak et al., 1998), as the function of VR1 as a nociceptor in the brain is rather unlikely, and instead a possible role in ligand-activated neurotransmitter modulation could be proposed (Szallasi and Di Marzo, 2000). Indeed, recent investigations have shown that VR1 activation in the brain leads to glutamate release in the locus coeruleus and substantia nigra (Marinelli et al., 2002, 2003), as well as in the periaqueductal gray (Palazzo et al., 2002; McGaraughty et al., 2003).

The chemical similarity between AEA and capsaicin, both of these two compounds being fatty acid amides, suggested that they might have a molecular target in common. The observation that the C18:1 capsaicin analog, olvanil (Figure 6.2) (Dray, 1992), inhibits AEA cellular uptake via the anan-damide membrane transporter (AMT) (Di Marzo et al., 1998; Beltramo and Piomelli, 1999) strengthened this suggestion. Based on this finding, an AEA/capsaicin structural "hybrid" named arvanil (Melck et al., 1999) (Figure 6.2), was synthesized and found to act as a partial agonist at CB1 receptors and a full agonist at VR1 receptors, and to be one of the most potent AMT inhibitors developed to date. These data suggested the existence of a partial overlap of the ligand recognition properties of VR1 and CB1 receptors and of VR1 and the AMT. Indeed, subsequent studies showed that AEA could produce a typical capsaicin-induced and calcitonin gene-related peptide (CGRP)-mediated response, i.e., the

Arvanil

Arvanil

FIGURE 6.2 Chemical structures of (1) some VR1 receptors agonists, (2) CB^VRl "hybrid" agonists, and (3) proposed ligands of non-CB^ non-VRl receptors in the brain.

relaxation of rodent small arteries in vitro, via a mechanism that was CBr and endothelium-independent and blocked by previous treatment of arteries with capsaicin and by the VR1 antagonist, capsazepine (Zygmunt et al., 1999). Accordingly, AEA activated the recombinant rat VR1 receptor overexpressed in either human embryonic kidney (HEK-293) cells or Xenopus oocytes, although less potently than with native vanilloid receptors in the rat mesenteric artery (Zygmunt et al., 1999). The subsequent discovery that a similar action was exerted more efficaciously and potently also at recombinant human VR1 receptors (Smart et al., 2000) triggered a series of studies aimed at investigating whether other pharmacological activities of this compound might be mediated by vanilloid receptors. There are now tens of reports indicating that AEA might work as an activator of native vanilloid receptors and possibly as an "endovanilloid," under both physiological and, particularly, pathological conditions, in vitro and in vivo. Neonatal AEA treatment can even mimic the prolonged mitochondrial damage of VR1-containing trigeminal neurons caused by capsaicin (Szoke et al., 2002). In peripheral sensory neurons, AEA induces the release of either tachykinins or CGRP or both, and causes diverse effects, including:

• Relaxation of small arteries (Zygmunt et al., 1999), particularly of the mesenteric artery, where most studies have been performed (Vanheel and Van de Voorde, 2001) but also of rabbit aortic rings (Ho and Hiley, 2003b; Harris et al., 2002; Mukhopadhyay et al., 2002; Ralevic et al., 2002 for review); these effects, or most likely the VR1-mediated stimulation of vagal fibers (see the following text) might be responsible for the transient, initial phase of the hypotensive effects of AEA in vivo in anesthetized normal and hypertensive rats (Malinowska et al, 2001; Li et al., 2003). By contrast, White et al., (2001) found that AEA relaxes the serotonin-precontracted rat coronary artery via a non-VR1, non-CB1 mechanism, even though the same authors confirmed that AEA relaxes the rat mesenteric artery via VR1 receptors and at low concentrations (EC50 = 200 nM).

• Stimulation of pulmonary vagal C-fibers in rats and guinea pigs (Lin and Li, 2002; Kagaya et al., 2002; Undem and Kollarik, 2002), or the stimulation of neurons of the nucleus solitary tract (Geraghty and Mazzone, 2002). The effects are likely to cause the cardiovascular and respiratory reflexes consisting, for example, of a fall in blood pressure and an increase in ventilation, observed after AEA injection intra-arterially in the hind limb of anesthetized rats (Smith and McQueen, 2001).

• Contraction of guinea pig bronchi, an effect that independent studies from many different laboratories (Tucker et al., 2001; De Petrocellis, Harrison, et al., 2001; Craib et al., 2001) have ascribed to activation of vanilloid receptors and which leads to cough (Jia et al., 2002).

• Inhibition of the contraction induced by endogenously released acetylcholine, or stimulation of neuropeptide release, in rat tracheae (Nemeth et al., 2003; Nieri et al., 2003). Interestingly, the latter can be observed only at higher doses, whereas an inhibitory effect due to CBj receptors is observed at lower doses (Nemeth et al., 2003).

• Relaxation of electrically-stimulated mouse vas deferens (Ross et al., 2001), an effect that is, in part, due also to a CB j-receptor-mediated mechanism; the involvement of sensory neuropeptides in this case was not investigated.

• Constriction of guinea pig ileum, an action likely to be mediated by an increase of acetylcholine release from myenteric neurones (Mang et al., 2001).

AEA stimulates the release of the sensory neuropeptides CGRP and substance P, also from rat dorsal root ganglia (DRG) (Tognetto et al., 2001). Whereas in the case of perivascular sensory neurons, AEA is quite potent in its action (Zygmunt et al., 1999), in the case of DRG neurons, however, the effect is seemingly observed only at high concentrations, and lower doses of AEA appear to be sufficient to exert a CB1-mediated inhibitory action on neuropeptide release (Tognetto et al., 2001). Very probably, for this reason a stronger stimulatory action, comparable in both potency and efficacy to the CB1-mediated inhibitory one, is instead observed with AEA when these experiments are performed in the presence of the CB1 antagonist SR141716A (Ahluwalia, Yaqoob, et al., 2003). These findings show good correlation with electrophysiological and in vivo studies where AEA activates nociceptive afferents innervating rat knee joints in both normal and arthritic animals, at high nanomolar doses (up to 800 nmol) (Gauldie et al., 2001), whereas it inhibits spinal neuronal responses in noninflamed and carrageenan-inflamed rats, at lower doses (up to 144 nmol) (Harris et al., 2000). Thus, while stimulation of CB receptors by synthetic agonists with no action on VR1 can lead to inhibition of capsaicin nociceptive and inflammatory effects (Richardson etal., 1998; Rukwied etal., 2003), exogenous AEA can induce either pro- or antinociceptive effects, depending on its concentration and, possibly, on the presence or absence of cannabinoid CB receptors on sensory neurons. Also, if one looks at the currents induced by AEA via VR1 in sensory and brain neurons, the potency for this effect is usually 1 to 2 orders of magnitude lower than that of capsaicin (Roberts etal., 2002; Marinelli etal., 2003; Jennings etal., 2003). However, this is not always true, because some DRG neuron preparations appear to respond to low concentrations of AEA only with VR1-mediated hyperactivity (Greffrath et al., 2002). Also the VRl-mediated effects of AEA on cytosolic calcium can be exerted at concentrations much lower than 1 u.l/, depending on the experimental conditions (see the following text). For example, a recent study (Hermann etal., 2003) showed that if CB and VR1 receptors are overexpressed in HEK cells (1) pre-stimulation with a CB agonist leads to a significant twofold potentiation of capsaicin effect on intracellular calcium, and (2) AEA is more potent in these cells as a VR1 agonist than in cells only expressing VR1 receptors. This system may have a physiological correspondent in DRG neurons, where CB and VR1 receptors are colocalized to some extent, not only when the neurons are in culture (Ahluwalia et al., 2000; Farquhar-Smith et al., 2000) but also in normal tissue (Bridges et al., 2003).

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