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. 2002 Jun 11;99(12):8400-5.
doi: 10.1073/pnas.122196999.

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

Susan M Huang  1 , Tiziana Bisogno, Marcello Trevisani, Abdulmonem Al-Hayani, Luciano De Petrocellis, Filomena Fezza, Michele Tognetto, Timothy J Petros, Jocelyn F Krey, Constance J Chu, Jeffrey D Miller, Stephen N Davies, Pierangelo Geppetti, J Michael Walker, Vincenzo Di Marzo
Affiliations

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

Susan M Huang et al. Proc Natl Acad Sci U S A. .

Abstract

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 (EC(50) approximately 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 (EC(50) = 1.5 +/- 0.3 microg). 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.

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Figures

Figure 1
Figure 1
Chemical structures of capsaicin, NADA, and O-methyl-NADA.
Figure 2
Figure 2
Identification and biosynthesis of NADA in brain. (a) Identical mass spectra of NADA and material in bovine striatal extract. Standard and extract were analyzed in negative-ion, product-ion scanning mode by using a triple-quadrupole mass spectrometer. (b) Purified extract of bovine striatum and synthetic NADA were treated with acetic anhydride to form N-arachidonoyl-3,4-diacetoxyphenylethylamine (di-acetylNADA). Multiple-reaction monitoring on a triple-quadrupole mass spectrometer revealed coeluting peaks with molecular/fragment ions 524/196 atomic mass units. (c) Quadrupole time-of-flight MS analysis of material in the brain extract in positive-ion mode yielded a mass estimate of 440.3189, which is within 4.6 ppm of the mass of NADA. Exact masses of fragment ions permitted reconstruction of NADA as shown in d. (e) Distribution of NADA (pmol/g wet weight tissue, means ± SEM, n = 4–12) in central and peripheral nervous system. hippoc., hippocampus; cerebell., cerebellum; thal., thalamus, mid., midbrain. (f) Biosynthesis of [3H]NADA from [3H]arachidonic acid (50 μM) and tyrosine (50 μM) in rat brain homogenates. Lipids from different incubates (boiled homogenate, enzymatically active homogenate, and homogenate in the presence of the catechol-O-methyltransferase inhibitor OR-486 at 30 μM (a concentration that blocks the conversion of tyrosine into dopamine) were extracted and purified by thin layer chromatography. A peak sensitive to OR-486 with the same Rf as synthetic NADA was scraped from the plate and reanalyzed under different eluting conditions (Inset). Similar results (except for the effect of OR-486) were obtained when incubating [3H]arachidonic acid (50 μM) and dopamine (50 μM). The data are representative of three experiments.
Figure 3
Figure 3
NADA activates peripheral VR1. (a) Effect of increasing concentrations of NADA and anandamide on intracellular Ca2+ in neonatal DRG neurons. *, P < 0.05 vs. NADA (t test). (b) Inhibition by VR1 antagonists of NADA (1 μM) or capsaicin (1 μM) enhancement of intracellular Ca2+ in isolated DRG neurons. *, P < 0.05 vs. vehicle (t test). (c) Effect of increasing concentrations of NADA and anandamide on the release of CGRP-LI and SP-LI from rat dorsal spinal cord slices. (d) Effect of capsaicin pretreatment, VR1 antagonism, and incubation in a Ca2+-free medium on the effect of NADA on CGRP-LI and SP-LI. *, P < 0.05 vs. vehicle (t test). ND, not detectable.
Figure 4
Figure 4
NADA induces thermal hyperalgesia in mice via VR1. (a) The effect of NADA was dose-dependent. (b) Time response of the effect of NADA (5 μg, administered into the plantar surface of a hind paw) and its blockade by the VR1 antagonists capsazepine (CPZ) or iodo-resiniferatoxin (IRTX), administered 10 min before NADA (data are means ± SEM of n = 50 for NADA alone, n = 19 for NADA+CPZ, and n = 15, for NADA+IRTX).
Figure 5
Figure 5
NADA activates VR1 and enhances paired-pulse depression in the rat hippocampal slice. (a) Example of synaptic responses recorded from a single slice stimulated with paired pulses at an interstimulus interval of 20 ms. The response on the Left was recorded under control conditions, and the one on the Right was recorded after perfusion with 1 μM NADA for 20 min. Note no change in PS1 but a decrease in the amplitude of PS2. (Scale bar, 2 mV and 10 ms.) The histogram shows the effect of NADA over a wider range of interpulse intervals. Open bars show control paired-pulse depression, and filled bars show that recorded after perfusion with 1 μM NADA. Bars represent mean ± SEM (n = 5), and * indicates a significant difference (P < 0.05, paired t test) from control at that specific interpulse interval. (b) Effect of 1 μM anandamide perfused for 20 min. (Scale bar, 1 mV and 10 ms.) (c) The summary histograms represent the change in PS2 amplitude evoked by 0.2 (n = 3) and 1 (n = 5) μM NADA, 1 and 10 μM anandamide (n = 4), 1 μM capsaicin (n = 5), 10 μM capsazepine (n = 3), 1 μM NADA in the presence of capsazepine (CAPZ, n = 3), 10 nM iodo-resiniferatoxin (I-RTX, n = 3), and 1 μM NADA in the presence of iodo-resiniferatoxin (n = 3). The values shown for 1 μM capsaicin and 10 μM anandamide are from ref. .
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