Neurosaente Vol. Sl, No. 3, pp. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 747-157, 1993 Printed in Great Britain 0306.4522/93 $6.00 + 0.00 Pergamon Press Ltd 8 1993 IBRO zyxwvutsrqpo CHARACTERIZATION OF RESINIFERATOXIN BINDING SITES ON SENSORY NEURONS: CO-REGULATION OF RESINIFERATOXIN BINDING AND CAPSAICIN SENSITIVITY IN ADULT RAT DORSAL ROOT GANGLIA J. WINTER*, C. S. J. WALPOLE, S. BEVAN and I. F. JAMES* Sandoz lnstitutc for Medical Research, 5 Gower Place, London WCIE 6BN, U.K. Abstract-Binding of [‘Hlresiniferatoxin was seen by autoradiography in sections of rat dorsal root ganglia and the superficial dorsal horn of the spinal cord. Membranes from rat dorsal root aanglia and spinal cord, but not other tissues, had saturable high-affinity binding sites for [3H]msinifcratokr. A series of capsaicin analogues competed for these sites. The sites probably correspond to capsaicin receptors. Systemic pretreatment of rats with capsaicin caused loss of capsaicin sensitivity in sensory neurons and a reduction in binding of resiniferatoxin to rat dorsal root ganglia, measured by binding assays and antoradiography. Adult rat dorsal root ganglion neurons cultured without nerve growth factor also lost their ~psaic~n-~nsitivity and showed reduced ~sinifemtox~n binding. Therefore, capsaicin responses in sensory zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF neurons may be regulated by nerve growth factor through control of the number of capsaicm receptors. Capsakin, an excitotoxic, pain-producing component of hot zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Capsicum peppers, is a useful tool for the study of nociceptive sensory neurons (for reviews, see Refs 2, 4, 10, 16, 18, 21, 30). Since exposure to capsaicin can cause a functional desensitization to subsequent noxious stimuli, capsaicin, or its analogues, may also be useful for alleviating some painful conditions.~,z’*35 The excitatory actions of capsaicin are due to its ability to open a nonselective cation channel in the sensitive neurons.‘.2 Until recently identification of capsaicin-sensitive neurons relied on functional assays based on evoked ion fluxes, electrophysio- logical measurement of capsaicin-induced membrane currents,2 accumulation of cobalt or 45Ca 1ons,34J8 or visual assessment of neurotoxic damage.36 There is indirect evidence that capsaicin acts on sensory neurons through a specific receptor linked to the capsaicin-sensitive ion channels. (1) There are fairly strict structural requirements for capsaicin-like activity (e.g. Ref. 31). (2) Capsaicin acts on mam- malian sensory neurons and not other cell types3* (3) Capsaicin sensitivity of cultured sensory neurons *To whom correspondence should be addressed. Abbreviations: APES, aminopropyltriethoxy-silane; BSA, bovine serum albumin; BSS, balanced salt solution; DRG. dorsal root ganglia; DMSO, dimethylsulphoxide; EDTA, ethylenediaminetetra-acetate; EGTA. ethylene- glycolbis (aminoetheylether) tetra-acetate; HPLC, high- performance liquid chromatography; NGF, nerve growth factor: ROPA. resiniferanol9.13.14-orthonhenvl- acetate; RTX, resinifcratoxin; SCG, superior cervical ganglion; SDS, sodium dodecyl suiphate; US-G, UltraserG; VAH, ~-vanillyl~6-aminohexamide; VBT, ~-vanillyl-ff’~butylthiourea; VOS. ~-vanil~yl-octyl- su~phona~de; VOT, ~-vanillyl-~-~tylthio~ea. is regulated by nerve growth factor (NGF35). (4) Photoaffinity probes based on capsaicin cause long- lasting agonist effects on sensory neurons.‘7,39 Until recently, direct identification of the receptor site in binding assays has not been possible. Capsaicin or dihydrocapsaicin were not suitable radioligands, because their relatively low potency,” and their l~pophilic nature meant that levels of non-saturable binding were high. The discovery that the highly potent irritant, resiniferatoxin (RTXY~i4*rS), mimicked some of the actions of capsaicin in viuo’ has made direct demon- stration of capsaicin binding sites possible. RTX is a diterpene found in some plants of the genus Euphorbiu. It has structural similarities to capsaicin and phorbol esters;9 but unlike phorbol esters, RTX is a poor activator of brain protein kinase C prep- arations,* and lacks tumour-promoting activity.42 RTX acts on the same cells via the same cellular mechanisms as capsaicin but is at least 100 times more potent in most tissues.‘.2S~M Szallasi and Blumber~7 were able to demonstrate specific bind- ing sites for 13H]RTX on membranes isolated from spinal cord and dorsal root ganglia (DRG) of rat and pig. The structure/activity requirements for binding to the RTX site suggest that it represents the capsaicin receptor.‘6-28 NGF regulates the sensitivity of cultured adult rat DRG neurons to capsaicin. Removal of NGF from the culture medium leads to loss of capsaicin sensi- tivity3’ without killing the neurons” since responses to capsaicin can be restored by replenishing the NGF in the culture medium for a few days. The available evidence suggests that the primary action of capsaicin is probably not dependent on any second messenger