The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKC is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKC and PKC␤1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKC enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKC reduced the response of rVR1. 2) Reduction of endogenous PKC using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKC localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKC directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKC and rVR1. 5) PKC directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKC and the enhancement by PKC of the rVR1 response to capsaicin. We conclude that PKC functions as a direct modulator of rVR1.The vanilloid receptor type 1 (VR1 or TRPV1)1 is a vanilloidgated, nonselective cation channel that belongs to the transient receptor potential (TRP) channel superfamily. VR1 is expressed on small diameter neurons within sensory ganglia and accounts for the highly selective action of vanilloids as excitatory agents for nociceptors. In addition to vanilloids, heat and protons also influence vanilloid receptors and nociceptive pathways, and VR1 thus can be viewed as a molecular integrator of chemical and physical stimuli that elicit pain (1). Following tissue injury, the magnitude of VR1 responses is modulated by the combined effects of protons, temperature, endogenous ligands, and signaling pathways, and this modulation of VR1 activity contributes to the sensitization of nociceptors associated with the development of allodynia and hyperalgesia (2).Current evidence implicates multiple signaling pathways in the modulation of VR1. PKA reduces vanilloid receptor type 1 (VR1) desensitization and directly phosphorylates VR1 (3). PKC␣ contributes to VR1 activation by pH (4), and PKC⑀ both directly phosphorylates VR1 and is implicated in the development of hyperalgesia (19). Finally, p38 mitogen-activated ...

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K(i) value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-2-benzylpropyl moieties in the C-region favored agonism.

The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-NЈ-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tertbutylbenzyl)-NЈ-[3-fluoro-4-(methylsulfonylamino)benzyl]-thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [ 3 H]resiniferatoxin binding to rVR1 with an affinity of 53.5 Ϯ 6.5 nM and antagonized capsaicininduced calcium uptake with an EC 50 of 9.2 Ϯ 1.6 nM, reflecting 25-and 60-fold greater potencies than capsazepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (Ͻ5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different activators could thus be differentially affected by different ligands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking response to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine.A vanilloid receptor (VR1) that is activated by capsaicin, low pH, and temperatures higher than 42°C has been cloned from rat dorsal root ganglia (Caterina et al., 1997;Tominaga et al., 1998). It is a nonselective cation channel, with high permeability for divalent cations, expressed on unmyelinated pain-sensing nerve fibers (C-fibers) and small A␦ fibers in the dorsal root, trigeminal, and nodose ganglia. Initially, activation of VR1 by pungent agonists such as capsaicin leads to excitation of primary sensory neurons gating nociceptive inputs to the central nervous system. Subsequently, these fibers may become desensitized/defunctionalized, and this desensitization forms a basis for the therapeutic use of VR1 agonists . Potential therapeutic applications include detrusor hyperreflexia, postherpetic neuralgia, diabetic neuropathy, cluster headache, osteoarthritis, and pruritus (Rains and Bryson, 1995;Kim and Chancellor, 2000).The exciting potential therapeutic applications for vanilloids have motivated efforts to identify or design novel derivatives with improved properties (Walpole et al., 1993a,b,c;Wrigglesworth et al., 1996). An important advance was the identification of resiniferatoxin (RTX), a diterpene related to the phorbol...