The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of ''hot'' chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous ''capsaicin-like'' substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 Ϸ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 ‫؍‬ 1.5 ؎ 0.3 g). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors. V anilloid receptors of type 1 (VR1) are nonselective cation channels, expressed in peripheral sensory C and A␦ fibers and gated by nociceptive stimuli such as low pH, heat, and some plant toxins, of which capsaicin, the pungent principle of chili peppers, is the best known example (1-4). Evidence obtained by several laboratories and using different techniques (5-10) showed that VR1 is present also in the central nervous system, where it is unlikely to be the target of noxious heat and low pH, thus suggesting the existence of brain endogenous agonists for this receptor (11). Indeed, lipid mediators previously known to serve other functions in the brain, i.e., the endocannabinoid anandamide and some lipoxygenase derivatives, activate VR1, albeit with a potency considerably lower than that of capsaicin (12)(13)(14). The antinociceptive effects of VR1 blockers in two models of inflammatory hyperalgesia (15, 16) suggest that ''endovanilloids'' might be produced also by peripheral tissues and act in concert with locally enhanced temperature and acidity during inflammation.If an endovanilloid did exist, what would be the structural prerequisites that would allow for an optimal interaction with vanilloid receptors? Structure-activity relationship studies for vanilloid receptors have indicated that both the vanillyl-amine moiety and a long, unsaturated acyl chain are necessary to...

N-Arachidonoyldopamine (NADA) was recently identified as an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicated the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Quadrupole time-of-flight mass spectrometric analysis of bovine striatal extracts revealed the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA failed to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there was no evidence of spontaneous pain behavior. By contrast, OLDA induced calcium influx (EC 50 ‫؍‬ 36 nM), reduced the latency of paw withdrawal from a radiant heat source in a dose-dependent manner (EC 50 ‫؍‬ 0.72 g), and produced nocifensive behavior. These effects were blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin (10 nM for HEK cells and 1 g/50 l for pain behavior). These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA was only a weak ligand for rat CB1 receptors; but like NADA, it was recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamine indicated the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors.We recently identified N-arachidonoyldopamine (NADA) 1 as an endogenous compound that possesses nanomolar potency for vanilloid type 1 (VR1) and cannabinoid CB1 receptors (1-3). Following injection into skin, NADA produces VR1-mediated thermal hyperalgesia, with an EC 50 of 1.5 g. Previous studies indicated that anandamide and the lipoxygenase product 12(S)-hydroperoxyeicosatetraenoic acid also possess affinity for VR1 (4, 5). However, whereas NADA is similar in potency to capsaicin in a variety of assays of VR1 activity, anandamide and 12(S)-hydroperoxyeicosatetraenoic acid are at least 20-fold less potent than capsaicin (4).All of the putative endovanilloids identified to date are products of arachidonic acid, an unsaturated long chain fatty acid with a primary role in inflammation and pain. Research on the structure-activity relationship for VR1 has shown that a vanillylamine moiety and a long unsaturated acyl chain are required for optimal interaction with the receptor (6, 7). Although anandamide and lipoxygenase products contain the long unsaturated acyl chain, they lack the vanillyl group, which probably accounts for their decreased affinity for and efficacy on VR1 compared with NADA. Because dopamine is a naturally occurring aromatic amine structurally similar to vanillylamine, it was hypothesized that certain unsaturated N-acyldopamines may exist i...

1 Four long-chain, linear fatty acid dopamides (N-acyldopamines) have been identified in nervous bovine and rat tissues. Two unsaturated members of this family of lipids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine, were shown to potently activate the transient receptor potential channel type V1 (TRPV1), also known as the vanilloid receptor type 1 for capsaicin. However, the other two congeners, N-palmitoyl-and N-stearoyl-dopamine (PALDA and STEARDA), are inactive on TRPV1. We have investigated here the possibility that the two compounds act by enhancing the effect of NADA on TRPV1 ('entourage' effect). 2 When pre-incubated for 5 min with cells, both compounds dose-dependently enhanced NADA's TRPV1-mediated effect on intracellular Ca 2 þ in human embryonic kidney cells overexpressing the human TRPV1. In the presence of either PALDA or STEARDA (0.1-10 mM), the EC 50 of NADA was lowered from B90 to B30 nM. 3 The effect on intracellular Ca 2 þ by another endovanilloid, N-arachidonoyl-ethanolamine (anandamide, 50 nM), was also enhanced dose-dependently by both PALDA and STEARDA. PALDA and STEARDA also acted in synergy with low pH (6.0-6.7) to enhance intracellular Ca 2 þ via TRPV1. 4 When co-injected with NADA (0.5 mg) in rat hind paws, STEARDA (5 mg) potentiated NADA's TRPV1-mediated nociceptive effect by significantly shortening the withdrawal latencies from a radiant heat source. STEARDA (1 and 10 mg) also enhanced the nocifensive behavior induced by carrageenan in a typical test of inflammatory pain. 5 These data indicate that, despite their inactivity per se on TRPV1, PALDA and STEARDA may play a role as 'entourage' compounds on chemicophysical agents that interact with these receptors, with possible implications in inflammatory and neuropathic pain.

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