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Endocannabinoids and Vanilloid TRPV1 Receptors

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Endocannabinoids and Vanilloid TRPV1 Receptors Endocannabinoids and vanilloid TRPV1 receptors Ruth A. Ross School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Forester- hill, Aberdeen AB25 2ZD, Scotland, UK Anandamide – endocannabinoid and endovanilloid? The endocannabinoid system comprises two known receptors (CB1 and CB2), a fam- ily of endogenous ligands, and specific molecular machinery for the synthesis, trans- port, and inactivation of cannabinoid ligands [1]. N-Arachidonoyl-ethanolamide (anandamide) (Fig. 1) is known as an endocannabinoid, as defined by its ability to be produced endogenously and to bind to and activate cannabinoid CB1 and CB2 receptors (see [1]). Since its discovery, anandamide has been shown to have numer- ous physiological actions that encompass cardiovascular, immune, gastrointestinal, and nervous systems [2–5]. The pharmacology of anandamide is complex, its actions being mediated by cannabinoid CB1 and CB2 receptors and by putative non- CB1, non-CB2 receptors [1, 6, 7]. The search for endogenous TRPV1 receptor acti- vators or endovanilloids is on-going and recent advances suggest that anandamide may be one such compound [8–11]. There is evidence that anandamide can activate TRPV1 receptors both directly and indirectly via lipoxygenase metabolites [12–15]. There is ample evidence that the interaction of anandamide with TRPV1 receptors is specific: TRPV1 actions are blocked by receptor- specific antagonists and not by antagonists of CB1 and CB2 receptors; desensitization of TRPV1 via capsaicin pre- treatment abrogates the effects of anandamide; neonatal capsaicin treatment pre- vents anandamide activation of TRPV1; anandamide-mediated TRPV1 effects are absent from untransfected cells that do not express TRPV1 receptors; and anan- damide displaces radiolabelled resiniferatoxin (RTX) from specific binding sites [12–14]. Whereas anandamide has similar affinity for TRPV1 to that of capsaicin, it has significantly lower potency [14]. In high-expression recombinant cell lines using var- ious methods, the potency (EC50) of anandamide has been measured in the range of 0.7–5 µM. In native systems, the potency of anandamide ranges from 0.3 to 0.8 µM in blood vessels (relaxation), compared with 6–10 µM in bronchus (contraction), dorsal root ganglion (DRG) neurons and trigeminal neurons (intracellular Ca2+ con- Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation, edited by Annika B. Malmberg and Keith R. Bley © 2005 Birkhäuser Verlag Basel/Switzerland 71 Ruth A. Ross Figure 1 Structures of arachidonic acid, anandamide and N-arachidonoyl-dopamine (NADA). 2+ centration, [Ca ]i, and inward current), and dorsal horn neurons (miniature exci- tatory postsynaptic currents (mEPSCs)) [16, 17]. Significant differences emerge when comparisons are made of the efficacy of anandamide in native and recombi- nant receptor systems. There are also significant differences between tissues. Using 2+ measurement of [Ca ]i in high-expression recombinant cell lines expressing either rat or human TRPV1, anandamide appears to be a full agonist, as defined by an Emax value that is not significantly different from that of capsaicin [13, 14, 18–20]. Some authors find the Emax value of anandamide to be less than 100% in recombi- nant expression systems. Similar differences are found in tissues expressing native TRPV1 receptors and the efficacy of anandamide appears to be tissue-dependent. Thus, it is a full agonist in blood vessels and mesenteric arterial bed, and a partial agonist in the bronchus, DRG, trigeminal neurons, and substantia gelatinosa neu- rons of the dorsal horn [13, 17, 21, 22]. Ross et al. [14] found that, although anan- 72 Endocannabinoids and vanilloid TRPV1 receptors damide has an Emax value of 100%, it displays a low Ki/EC50 ratio, as determined from radioligand binding and 45Ca2+-uptake data; this is indicative of low intrinsic efficacy. As a consequence of the low intrinsic efficacy of anandamide at TRPV1, the Emax value will vary with the receptor reserve and the pharmacological endpoint 2+ that is measured. Thus, whereas elevation of [Ca ]i has a high degree of signal amplification, measurement of TRPV1 current has no downstream amplification. The low intrinsic efficacy of anandamide at the TRPV1 receptor has important physiological implications. A low intrinsic efficacy agonist will attenuate the effects of a full agonist. Indeed, in trigeminal neurons, co-application of anandamide with capsaicin significantly reduces the currents produced by capsaicin [22]. If endoge- nous activators of TRPV1 exist that are full agonists, then compounds such as anan- damide may have an inhibitory role and serve as anti-inflammatory and analgesic compounds. Intracellular versus extracellular It is important to give consideration to the location of the agonist-binding site on TRPV1, which is located intracellularly [23, 24]: the apparent low intrinsic effi- cacy of anandamide may be due to limited access to this binding site. As a conse- quence, the potency of exogenously applied anandamide will be affected by its ability to enter the cell (see [16]). Anandamide gains access to the intracellular environment either via the anandamide membrane transporter (AMT) or via accu- mulation sustained by a concentration gradient that is maintained through the rapid intracellular hydrolysis of anandamide by fatty acid amide hydrolase (FAAH; see [25, 26]). The finding of the enhanced potency of anandamide when administered intracellularly is in line with the demonstration that compounds that alter the function of the AMT modulate the potency of anandamide in HEK-293 cells expressing TRPV1 [27–29]. A recent study by Andersson et al. [21] provides further evidence that primary afferent fibers express the AMT and that variability in the expression levels of the transporter affect the potency of anandamide. Thus the AMT inhibitor, VDM13, causes a 2.3-fold rightward shift in the log concen- tration-response curve for vasodilatation of mesenteric arteries by anandamide but does not affect the contractile response to anandamide in the bronchus. Similarly, the potency of the putative endovanilloid N-arachidonoyl-dopamine (NADA; Fig. 1) varies between tissue preparations, an observation that has been attributed to differential expression of the AMT [30, 31]. As has been demonstrated that for CB1 receptors [32] the affinity and potency of anandamide at TRPV1 is enhanced by inhibition of FAAH in certain tissues, including Chinese hamster ovary cells [14], HEK-293 cells, rat ileum, and neuroblastoma cells (see [16]). On the other hand, in the mesenteric bed, FAAH inhibitors attenuate the TRPV1 receptor-medi- ated relaxation by anandamide [33]. In the bronchus and trigeminal neurons, 73 Ruth A. Ross however, the potency of anandamide as a TRPV1 agonist is unaffected by FAAH inhibitors [21, 34, 35]. Recent data represent the first direct demonstration of a significant difference between the potency of anandamide when the compound is applied directly to the intracellular environment (Fig. 2). As previously demonstrated by others [12, 13, 22, 25], Evans et al. [11] confirm that, when applied to the extracellular environ- ment, anandamide had low potency at the TRPV1 receptor in rat cultured DRG neurons. Consequently they found that, when applied extracellularly, 100 nM anan- damide fails to produce inward currents in 93% of neurons. In contrast, when applied intracellularly, this relatively low concentration evoked inward currents in 60% of neurons, the current being significantly attenuated by the TRPV1 receptor antagonist, capsazepine (1 µM). In another demonstration of intracellular activation of TRPV1, Hwang et al. [35] recorded TRPV1 receptor-mediated inward currents in inside-out patches of cultured DRG neurons: anandamide activated TRPV1 receptors, but the efficacy was found to be much lower than that of capsaicin. Dif- ferences in compound preparation and vehicles may account for the variation in potency observed. These data raise the possibility that anandamide may be a relatively high-poten- cy TRPV1 agonist when it is released intracellularly, close to the TRPV1 receptor ligand-binding site. Mass spectrometric analysis shows that both capsaicin and depolarization (KCl) induce significant release of anandamide in DRG cultures [2, 36]. It is also notable that the capsaicin-evoked release of anandamide is signifi- cantly attenuated when the FAAH inhibitor, methylarachidonyl-fluorophosphonate (MAFP), is excluded from the buffer, demonstrating that anandamide is rapidly metabolized in DRG neurons. It would therefore appear that anandamide can acti- vate both CB1 and TRPV1 receptors in a similar concentration range, raising the possibility that it is indeed both an endocannabinoid and an endovanilloid. CB1/TRPV1 crosstalk An intriguing possibility is that when applied to the extracellular environment, anandamide first activates CB1 receptors, whose ligand-binding domain is extracel- lular, which in turn modulates activation of TRPV1 (see [16, 37]). CB1/TRPV1 signaling Phosphorylation of TRPV1 by protein kinase C (PKC) and protein kinase A (PKA) sensitizes TRPV1 receptors to vanilloids, anandamide, heat, and protons (see [2]). In contrast, PKC-mediated phosphorylation of the CB1 receptor prevents cannabi- 2+ noid-mediated activation of Kir currents and inhibition of P/Q-type Ca channels 74 Endocannabinoids and vanilloid TRPV1 receptors Figure 2 Intracellular versus extracellular application of anandamide in dorsal root ganglion (DRG) neurons. Using
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