Pain is initiated when the peripheral terminals of a particular group of sensory neurons, called nociceptors, are activated by noxious chemical, mechanical, or thermal stimuli. These neurons, whose cell bodies are located in various sensory ganglia, transmit information regarding tissue damage to pain processing centers in the spinal cord and brain (Fields Pain (McGraw-Hill, New York, 1987)). Nociceptors are characterized, in part, by their sensitivity to capsaicin, a natural-product of capsicum peppers that is the active ingredient of many “hot” and spicy foods. In mammals, exposure of nociceptor terminals to capsaicin leads initially to the perception of pain and the local release of neurotransmitters. With prolonged exposure, these terminals become insensitive to capsaicin, as well as to other noxious stimuli (Szolcsanyi in Capsaicin in the Study of Pain (ed. Wood) pgs. 255–272 (Academic Press, London, 1993)). This latter phenomenon of nociceptor desensitization underlies the seemingly paradoxical use of capsaicin as an analgesic agent in the treatment of painful disorders ranging from viral and diabetic neuropathies to rheumatoid arthritis (Campbell in Capsaicin and the Study of Pain (ed. Wood) pgs. 255–272 (Academic Press, London, 1993); Szallasi et al. 1996 Pain 68:195–208). While some of this-decreased sensitivity to noxious stimuli may reflect reversible changes in the nociceptor, such as depletion of inflammatory mediators, the long-term loss of responsiveness can be explained by death of the nociceptor or destruction of its peripheral terminals following capsaicin exposure (Jancso et al. 1977 Nature 270:741–743; Szolcsanyi, supra).
Responsivity to capsaicin has been used to define sensory afferent fibers that transmit signals in response to noxious stimuli (chemical, thermal, and mechanical stimuli); however the precise mechanism of action has remained unclear. Electrophysiological (Bevan et al. 1990 Trends Pharmacol. Sci 11:330–333; Oh et al. 1996 J. Neuroscience 16:1659–1667) and biochemical (Wood et al. 1988 J. Neuroscience 8:3208–3220) studies have clearly shown that capsaicin excites nociceptors by increasing plasma membrane conductance through formation or activation of nonselective cation channels. While the hydrophobic nature of capsaicin has made it difficult to rule out the possibility that its actions are mediated by direct perturbation of membrane lipids (Feigin et al. 1995 Neuroreport 6:2134–2136), it has been generally accepted that this compounds acts at a specific receptor site on or within sensory neurons due to observations that capsaicin derivatives show structure-function relationships and evoke dose-dependent responses (Szolcsanyi et al. 1975 Drug. Res. 25:1877–1881; Szolcsanyi et al. 1976 Drug Res. 26:33–37)). The development of capsazepine, a competitive capsaicin antagonist (Bevan et al. 1992 Br. J. Pharmacol. 107:544–552) and the discovery of resiniferatoxin, an ultrapotent capsaicin analogue from Euphorbia plants that mimics the cellular actions of capsaicin (deVries et al. 1989 Life Sci. 44:711–715; Szallasi et al. 1989 Neuroscience 30:515–520) further suggest that the capsaicin mediates its effects through a receptor. The nanomolar potency of resiniferatoxin has facilitated its use as a high affinity radioligand to visualize saturable, capsaicin- and capsazepine-sensitive binding sites on nociceptors (Szallasi 1994 Gen. Pharmac. 25:223–243). Because a vanilloid moiety constitutes an essential structural component of capsaicin and resiniferatoxin, the proposed site of action of these compounds has been more generally referred to as the vanilloid receptor (Szallasi 1994 supra). The action of capsaicin, resiniferatoxin, and the antagonist capsazepine have been well characterized physiologically using primary neuronal cultures (see, e.g., Szolcsanyi, “Actions of Capsaicin on Sensory Receptors,” Bevan et al. “Cellular Mechanisms of the Action of Capsaicin,” and James et al. “The Capsaicin Receptor,” all in Capsaicin in the Study of Pain, 1993 Academic Press Limited, pgs. 1–26, 27–44, and 83–104, respectively; Bevan et al. 1990, supra).
The analgesic properties of capsaicin and capsaicinoids are of much interest for their uses in the treatment of pain and inflammation associated with a variety of disorders (see, e.g, Fusco et al. 1997 Drugs 53:909–914; Towheed et al. 1997 i. Arthritis Rheum 26:755–770; Appendino et al. 1997 Life Sci 60:681–696 (describing activities and application of resiniferatoxin); Campbell et al. “Clinical Applications of Capsaicin and Its Analogues” in Capsaicin in the Study of Pain 1993, Academic Press pgs. 255–272). Although capsaicin and capsaicin related compounds can evoke the sensation of pain, cause hyperalgesia, activate autonomic reflexes (e.g., elicit changes in blood pressure), and cause release of peptides and other putative transmitters from nerve terminals (e.g., to induce bronochoconstriction and inflammation), prolonged exposure of sensory neurons to these compounds leads to desensitization of the neurons to multiple modalities of noxious sensory stimuli without compromising normal mechanical sensitivity or motor function, and without apparent central nervous system depression. It is this final phenomena that makes capsaicins and related compounds of great interest and potential therapeutic value.
Despite the intense interest in capsaicin and related compounds and their effects upon sensory afferent, the receptor(s) through which these compounds mediate their effects have eluded isolation and molecular characterization. Thus, the development of elegant systems for screening or characterizing new capsaicin receptor-binding compounds, or for identifying endogenous, tissue-derived mediators of pain and/or inflammation, have been severely hampered. To date the only means of assessing the activity of compounds as capsaicin receptor agonists or antagonists has been to examined their effects on sensory neurons in culture or in intact animals. The present invention solves this problem.