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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/PKCmu 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 PKCmu and PKCbeta1, 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 PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu 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) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu 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 PKCmu and rVR1. 5) PKCmu 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 PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.

In a continuing effort to elucidate the structure-activity relationships of the lead antagonist N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea (1), the distances between the proposed four pharmacophores in 1 have been varied by insertion or deletion of one carbon to optimize their fit to the receptor. In addition, the acyloxy group of the C region was replaced with amide and N-hydroxy amide to identify the pharmacophoric importance of the ester group in the C2 region. The results indicated that the pharmacophoric arrangement of 1 was optimal for receptor binding affinity and antagonism, and the ester of the C2 region was significant for receptor binding. Among the derivatives, compound 19 showed distinct behavior with a 2-fold improvement in antagonism but a 13-fold reduction in binding affinity compared to 1. The partial separation of pharmacophoric requirements of these two assays has been noted before and compound 19 is thus selective for the calcium entry-linked receptor population. The conformational analysis of 1 generated three distinct conformers having different types of hydrophobic interactions, which will be utilized for exploring the active conformation of the VR1 ligand.

The vanilloid receptor (VR1) is a central integrator molecule of nociceptive stimuli. In this study, we have measured the effects of various neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and -4) on recombinant rat VR1-mediated intracellular calcium rise in response to capsaicin in VR1/C6 cells. Our results clearly show that all neurotrophins sensitize the VR1 to capsaicin. Furthermore, using K252a, an inhibitor of tyrosine kinases, we present that actions of neurotrophins are mediated by the trk (A, B, C) receptors expressed in these cells. These data argue for the putative roles of neurotrophins in inducing inflammatory (thermal) hyperalgesia via VR1.

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.

The structural modifications on the B-region of the potent and high affinity vanilloid receptor (VR1) lead ligand N-(3-acyloxy-2-benzylpropyl)-N(')-[4-(methylsulfonylamino)benzyl]thiourea were investigated by the replacement of the thiourea with diverse isosteric functional groups. Structure-activity analysis indicated that the A-region in this series was the primary factor in determining the agonistic/antagonistic activities regardless of the B-region. The N(C)-hydroxy thiourea analogues (12, 13) showed excellent analgesic activities in the acetic acid writhing assay compared to the parent thiourea analogues.

We previously described a series of N-(3-acyloxy-2-benzylpropyl) homovanillate and N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives that were potent VR1 agonists with high-affinities and excellent analgesic profiles. The design of these simplified RTX analogues was based on our RTX-derived pharmacophore model which incorporates the 4-hydroxy-3-methoxyphenyl (A-region), C(20)-ester (B-region), orthophenyl (C1-region) and C(3)-keto (C2-region) groups of RTX. For the purpose of optimizing the spatial arrangement of the four principal pharmacophores on the lead agonists (1-4), we have modified the distances in the parent C-region, 3-acyloxy-2-benzylpropyl groups, by lengthening or shortening one carbon to vary the distances between the pharmacophores. We find that two of the amides, 4 and 19, possess EC(50) values <1 nM for induction of calcium influx in the VR1-CHO cells. As observed previously, the structure-activity relations for inhibition of RTX binding to VR1 and for induction of calcium uptake were distinct, presumably reflecting both intrinsic and methodological factors. In order to find the active conformation of VR1 ligands, the energy-minimized conformations of seven selected agonists were determined and the positions of their four pharmacophores were matched with those of five low energy RTX conformations. The rms values for the overlaps in the pharmacophores were calculated and correlated with the measured binding affinities (K(i)) and calcium influx (EC(50)) values. The binding affinities of the agonists correlated best with the RMS values derived from RTX conformation E (r(2)=0.92), predicting a model of the active conformation of RTX and related vanilloids for binding to VR1. Poorer correlation was obtained between any of the conformations and the EC(50) values for calcium influx.

We describe the synthesis and characterization of N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (IBTU), a novel antagonist of the vanilloid receptor 1 (TRPV1 or VR1). IBTU competitively inhibited 45Ca2+ uptake into CHO cells heterologously expressing rat TRPV1, whether induced by capsaicin or resiniferatoxin (Ki = 99 +/- 23 and 93 +/- 34 nM, respectively). IBTU was thus somewhat more potent (5-fold) than capsazepine. In contrast to its antagonism of vanilloid-induced calcium uptake, IBTU (30 microM) inhibited [3H]resiniferatoxin binding to TRPV1 by less than 10%. We hypothesize that these dramatically distinct potencies reflect different fractions of TRPV1 in this system: namely, a minor plasma membrane fraction controlling 45Ca2+ uptake, and the predominant intracellular fraction that dominates the [3H]resiniferatoxin binding measurements. Intracellular Ca2+ imaging supports this explanation. IBTU antagonized the elevation in intracellular Ca2+ in response to 50 nM capsaicin with an IC50 of 106 +/- 35 nM. Likewise, 600 nM IBTU was able to antagonize the elevation in intracellular Ca2+ in response to 100 pM resiniferatoxin in the presence of normal (1.8 mM) extracellular Ca2+, where the increase in intracellular calcium reflects calcium influx. In contrast, in the absence of extracellular Ca2+, where in this system resiniferatoxin induces a modest increase in calcium from intracellular stores, IBTU was unable to block the response to resiniferatoxin, although the TRPV1 antagonist 5-iodoresiniferatoxin was able to do so. In summary, IBTU is a novel, potent TRPV1 antagonist with marked selectivity between subpopulations of TRPV1 and may permit the function of these distinct pools to be explored and potentially exploited.

We recently reported that N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl] thiourea (2) was a high affinity antagonist of the vanilloid receptor with a binding affinity of K(i)=63 nM and an antagonism of K(i)=53.9 nM in rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells (Mol. Pharmacol. 2002, 62, 947-956). In an effort to further improve binding affinity and antagonistic potency, we have modified the C-region of the lead 4-t-butylbenzyl group with diverse surrogates, such as araalkyl, alkyl, 4-alkynylbenzyl, indanyl, 3,3-diarylpropyl, 4-alkoxybenzyl, 4-substituted piperazine and piperidine. The lipophilic surrogates, arylalkyl and alkyl, conferred modest decreases in binding affinities and antagonistic potencies; the groups having heteroatoms resulted in dramatic decreases. Our findings indicate that 4-t-butylbenzyl is one of the most favorable groups for high receptor binding and potent antagonism to VR1 in this structural series.

In this study, we expressed rat vanilloid receptor 1 (VR1) in various heterologous expression systems using different VR1-encoding vectors, and examined how the VR1 agonists capsaicin and resiniferatoxin affected intracellular calcium. Our results clearly show that the magnitude and kinetics of response as well as the extent of tachyphylaxis differ markedly between systems. Using green fluorescent protein-tagged VR1, we show that much of the VR1 is localized to intracellular membranes. Consistent with this localization, VR1 agonists are able to liberate calcium from intracellular stores in the absence of extracellular calcium. As with other parameters of response, the three expression systems differ in the degree to which, in the absence of extracellular calcium, capsaicin and resiniferatoxin can liberate calcium from the intracellular stores. Our findings emphasize the influence of the expression system on characteristics of the response of VR1 to its ligands and the need for caution in extrapolating such results to other settings.

The vanilloid receptor VR1 is a polymodal nociceptor sensitive to capsaicin, protons, and heat. Because VR1 represents an attractive therapeutic target for conditions ranging from long-term pain to bladder hyperreflexia, we and other groups have sought to develop novel ligands with enhanced potencies and novel pharmacological properties. Here, we characterize two compounds, N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea (JYL827) and N-(4-tert-butylbenzyl)-N'-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea (JYL1511), that function as partial agonists for rat VR1 heterologously expressed in Chinese hamster ovary cells. Both compounds showed substantially enhanced potency, inhibiting [3H] resiniferatoxin binding with Ki values of 29.3 +/- 7.6 and 50.4 +/- 16.5 nM, respectively, compared with 1810 +/- 270 nM for capsaicin. The compounds showed different extents of partial agonism, 6.8 +/- 0.7% and 17.4 +/- 0.6%, respectively, and the expected corresponding degrees of partial antagonism (93.9 +/- 0.9 and 84.1 +/- 3.2%, respectively). Their IC50 values for antagonism of 45Ca2+ uptake in response to capsaicin were 67.3 +/- 24.9 nM and 3.4 +/- 0.5 nM, respectively. Protons, temperature, and protein kinase C all function as coactivators/modulators of rVR1. All enhanced the extent of partial agonism of JYL827 and JYL1511. Thus, at pH 5.5, for example, the extents of partial agonism increased to 54.9 +/- 2.5% and to 90.7 +/- 1.7%, respectively, relative to the response elicited by 300 nM capsaicin. The extents of partial antagonism decreased correspondingly. Compounds such as JYL827 and JYL1511 now permit exploration of the potential utility of partial agonists of rVR1 in animal models. Our results emphasize, moreover, the strong dependence of such partial agonists on other modulators of rVR1 and predict that their biological behavior will depend strongly on biological context.

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 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.

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)-N'-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tert-butylbenzyl)-N'-[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 +/- 6.5 nM and antagonized capsaicin-induced calcium uptake with an EC(50) of 9.2 +/- 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 (<5.5). 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.

Recent work has addressed the role of vanilloid receptor type 1 (VR1) in pain perception. VR1 activity is regulated both directly and indirectly by endogenous factors. For example, protein kinase C sensitizes human VR1 to mild decreases in pH, which are commonly encountered during inflammation, and renders the endocannabinoid anandamide a more potent 'endovanilloid'. Bradykinin and nerve growth factor release VR1 from the inhibitory control of phosphatidylinositol (4,5)-bisphosphate and anti-VR1 serum ameliorates thermal allodynia and hyperalgesia in diabetic mice. There is strong evidence that not only the sensitivity but also the density of expression of VR1 is enhanced during inflammatory conditions. These observations provide an empirical foundation which could explain the reduced inflammatory hyperalgesia in VR1 knockout mice, and they imply an important role for endovanilloid signaling via VR1 in the development of ongoing pain in humans that occurs in most inflammatory conditions. Conversely, downregulation of VR1 expression and/or activity is a promising therapeutic strategy for novel analgesic drugs.

We investigated the effect of thapsigargin, a well-known sarcoplasmic reticulum ATPase (SERCA) inhibitor, on the non-specific Ca2+ channel vanilloid receptor-1 (VR1) in CHO-VR1 cells. We found that thapsigargin inhibited the VR-1 mediated (45)Ca2+ uptake of CHO-VR1 cells (IC50=6.4+/-1.9 microM) and the [3H]RTX binding to VR1 (IC50=4.0+/-1.3 microM). Further analysis revealed that thapsigargin is a mixed-type inhibitor, suggesting both direct and indirect interactions between thapsigargin and the capsaicin binding site of VR1. Thapsigargin alone transiently elevated the [Ca2+]i in CHO-VR1 cells (EC50=44 nM). However, 45Ca2+ uptake was not detected after thapsigargin treatment, indicating that the emptying of the thapsigargin sensitive intracellular pools of Ca2+ was responsible for the elevated [Ca2+]i level rather than the activation of VR-1. We conclude that thapsigargin represents a new prototype of a VR1 inhibitor and that caution should be exercised in interpreting the effects of thapsigargin, especially when it is used in the micromolar range to inhibit SERCA activity., (Copyright 2002 Elsevier Science (USA).)

In order to improve the analgesic activity and pharmacokinetics of thioureas 2 and 3, which we previously developed as potent vanilloid receptor (VR) agonists, we prepared and characterized phenolic modifications of them and of their amide surrogates (7, 8). The aminoethyl analogue of the amide template 13 was a potent analgesic with an EC50=0.96 microg/kg in the AA-induced writhing test and with better in vivo stability than the parent phenol.

Specific [3H]resiniferatoxin (RTX) binding detects the vanilloid receptor type I (VR1). In the present study we demonstrate specific, high-affinity, saturable [3H]RTX binding in various areas of monkey brain not known to be innervated by primary afferent neurons as well as in spinal cord and dorsal root ganglion neurons of the same origin. Detailed pharmacological characterization and comparison revealed no major difference in binding affinities between the peripheral and the central sites as measured by K(d)/K(i) values. In general, lower receptor density was measured in selected brain areas than in the periphery. Areas with higher receptor density were detected in the locus ceruleus, preoptic area, and medial basal hypothalamus of the brain. Both capsaicin and the competitive antagonist capsazepine inhibited the specific binding of [3H]RTX to membrane preparations of the dorsal horn of the spinal cord and dorsal root ganglia with K(i) values of 4.3+/-0.32 microM and 2.7+/-0.33 microM, respectively. Inhibition was observed in the central areas (hypothalamus) with K(i) values of 0.95+/-0.1 microM for capsaicin and 0.86+/-0.11 microM for capsazepine. Previous biological and pharmacological evidence suggested that vanilloid receptors were present in the brain. Our results demonstrate that the pharmacological properties of both the peripheral and central receptor sites display appropriate pharmacological similarity to represent the same receptor class. The modest differences in ligand affinities for the vanilloid receptor expressed in the brain nuclei and the dorsal root ganglion neurons may correspond to differences in sequence, modification or associated proteins.

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [(3)H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [(3)H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

Since 1990, the National Cancer Institute has performed extensive in vitro screening of compounds for anticancer activity. To date, more than 70 000 compounds have been screened for their antiproliferation activities against a panel of 60 human cancer cell lines. We probed this database to identify novel structural classes with a pattern of biological activity on these cell lines similar to that of the phorbol esters. The iridals form such a structural class. Using the program Autodock, we show that the iridals dock to the same position on the C1b domain of protein kinase C delta as do the phorbol esters, with the primary hydroxyl group of the iridal at the C3 position forming two hydrogen bonds with the amide group of Thr12 and with the carbonyl group of Leu 21 and the aldehyde oxygen of the iridal forming a hydrogen bond with the amide group of Gly23. Biological analysis of two iridals, NSC 631939 and NSC 631941, revealed that they bound to protein kinase C alpha with K(i) values of 75.6 +/- 1.3 and 83.6 +/- 1.5 nM, respectively. Protein kinase C is now recognized to represent only one of five families of proteins with C1 domains capable of high-affinity binding of diacylglycerol and the phorbol esters. NSC 631939 and NSC 631941 bound to RasGRP3, a phorbol ester receptor that directly links diacylglycerol/phorbol ester signaling with Ras activation, with K(i) values of 15.5 +/- 2.3 and 41.7 +/- 6.5 nM, respectively. Relative to phorbol 12,13-dibutyrate, they showed 15- and 6-fold selectivity for RasGRP3. Both compounds caused translocation of green fluorescent protein tagged RasGRP3 expressed in HEK293 cells, and both compounds induced phosphorylation of ERK1/2, a downstream indicator of Ras activation, in a RasGRP3-dependent fashion. We conclude that the iridals represent a promising structural motif for design of ligands for phorbol ester receptor family members.

Vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that can be activated by capsaicin and other vanilloids as well as by protons and heat. In the present study, we have analyzed the oligomeric state of VR1. Co-immunoprecipitation of differently tagged VR1 molecules indicated that VR1 can form oligomers. Using two different heterologous VR1 expression systems as well as endogenous VR1 expressed in dorsal root ganglion cells, we analyzed oligomer formation using perfluoro-octanoic acid polyacrylamide gel electrophoresis. Results were confirmed both with chemical cross-linking agents as well as through endogenous cross-linking mediated by transglutaminase. Our results clearly show that VR1 forms multimers in each of the expression systems with a homotetramer as a predominant form. The oligomeric structure of VR1 may contribute to the complexity of VR1 pharmacology. Finally, differences in glycosylation between the systems were observed, indicating the need for caution in the use of the heterologous expression systems for analysis of VR1 properties.

The vanilloid receptor represents a promising target for drug development. Building on our previous strategies which have generated potent agonists for VR1, we now describe a series of novel N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues, several of which are potent VR1 antagonists. We report here the rationale for the design, the synthesis, and the in vitro characterization of activity in assays for [(3)H]resiniferatoxin binding and (45)Ca influx using heterologously expressed rat VR1.

The real time dynamics of vanilloid-induced cytotoxicity and the specific deletion of nociceptive neurons expressing the wild-type vanilloid receptor (VR1) were investigated. VR1 was C-terminally tagged with either the 27-kDa enhanced green fluorescent protein (eGFP) or a 12-amino acid epsilon-epitope. Upon exposure to resiniferatoxin, VR1eGFP- or VR1epsilon-expressing cells exhibited pharmacological responses similar to those of cells expressing the untagged VR1. Within seconds of vanilloid exposure, the intracellular free calcium ([Ca(2+)](i)) was elevated in cells expressing VR1. A functional pool of VR1 also was localized to the endoplasmic reticulum that, in the absence of extracellular calcium, also was capable of releasing calcium upon agonist treatment. Confocal imaging disclosed that resiniferatoxin treatment induced vesiculation of the mitochondria and the endoplasmic reticulum ( approximately 1 min), nuclear membrane disruption (5-10 min), and cell lysis (1-2 h). Nociceptive primary sensory neurons endogenously express VR1, and resiniferatoxin treatment induced a sudden increase in [Ca(2+)](i) and mitochondrial disruption which was cell-selective, as glia and non-VR1-expressing neurons were unaffected. Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory ganglia, may be employed to rapidly and selectively delete nociceptive neurons.

A series of N-(3-acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives were investigated as vanilloid receptor ligands in an effort to discover a novel class of analgesics. The proposed pharmacophore model of resiniferatoxin. which includes the C20 homovanillic moiety, the C3-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-inflammatory activity and pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among the new compounds, 23 and 28 were found to be the most potent receptor agonists of the series with Ki values of 19 nM and 11 nM, respectively. Their strong in vitro potencies were also reflected by an excellent analgesic profile in animal tests with ED50 values of 0.5 microg kg for 23 and 1.0 microg/kg for 28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both 23 and 28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show significant anti-inflammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and 35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in this investigation, which were designed as simplified resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor agonists endowed with potent analgesic activity and reduced pungency.

The cloned vanilloid receptor VR1 has attracted recent attention as a molecular integrator of painful stimuli on primary sensory neurons. The existence of vanilloid-sensitive neurons in the brain is, however, controversial. In this study, we have used an antibody and a complementary RNA probe to explore the distribution of neurons that express VR1 in rat and in certain areas of human brain. In the rat, we observed VR1-expressing neurons throughout the whole neuroaxis, including all cortical areas (in layers 3 and 5), several members of the limbic system (e.g., hippocampus, central amygdala, and both medial and lateral habenula), striatum, hypothalamus, centromedian and paraventricular thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, cerebellum, and inferior olive. VR1-immunopositive cells also were found in the third and fifth layers of human parietal cortex. Reverse transcription-PCR performed with rat VR1-specific primers verified the expression of VR1 mRNA in cortex, hippocampus, and hypothalamus. In the central nervous system, neonatal capsaicin treatment depleted VR1 mRNA from the spinal nucleus of the trigeminal nerve, but not from other areas such as the inferior olive. The finding that VR1 is expressed not only in primary sensory neurons but also in several brain nuclei is of great importance in that it places VRs in a much broader perspective than pain perception. VRs in the brain (and putative endogenous vanilloids) may be involved in the control of emotions, learning, and satiety, just to name a few exciting possibilities.

A series of 3-acyloxy-2-phenalkylpropyl amides and esters of homovanillic acid were designed and synthesized as vanilloid receptor agonists containing the three principal pharmacophores of resiniferatoxin. Amide analogues 23, 5 and 11 were found to be potent agonists in vanilloid receptor assay both for ligand binding and for activation.

1 Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities. 2 Phorbol 12,13-didecanoate 20-homovanillate (PDDHV) evoked 45Ca(2+)-uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]-resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin-type pharmacology. 3 45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors. 4 PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]-RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non-transfected CHO cells, confirming its action through VR1. 5 We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin-type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure-activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin-type vanilloid pharmacology.

[(3)H]Resiniferatoxin (RTX) binding and calcium uptake by rat dorsal root ganglion (DRG) neurons show distinct structure-activity relations, suggestive of independent vanilloid receptor (VR) subtypes. We have now characterized ligand binding to rat VR1 expressed in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells and compared the structure-activity relations with those for calcium mobilization. Human embryonic kidney cells (HEK293/VR1 cells) and Chinese hamster ovary cells transfected with VR1 (CHO/VR1 cells) bound [(3)H]RTX with affinities of 84 and 103 pM, respectively, and positive cooperativity (Hill numbers were 2.1 and 1.8). These parameters are similar to those determined with rat DRG membranes expressing native VRs (a K(d) of 70 pM and a Hill number of 1.7). The typical vanilloid agonists olvanil and capsaicin inhibited [(3)H]RTX binding to HEK293/VR1 cells with K(i) values of 0.4 and 4.0 microM, respectively. The corresponding values in DRG membranes were 0.3 and 2.5 microM. HEK293/VR1 cells and DRG membranes also recognized the novel vanilloids isovelleral and scutigeral with similar K(i) values (18 and 20 microM in HEK293/VR1 cells; 24 and 21 microM in DRGs). The competitive vanilloid receptor antagonist capsazepine inhibited [(3)H]RTX binding to HEK293/VR1 cells with a K(i) value of 6.2 microM and binding to DRG membranes with a K(i) value of 8.6 microM. RTX and capsaicin induced calcium mobilization in HEK293/VR1 cells with EC(50) values of 4.1 and 82 nM, respectively. Thus, the relative potencies of RTX (more potent for binding) and capsaicin (more potent for calcium mobilization) are similar in DRG neurons and cells transfected with VR1. We conclude that VR1 can account for both the ligand binding and calcium uptake observed in rat DRG neurons.

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. Here, we describe specific vanilloid responses in rat C6 glioma cells. Capsaicin and RTX stimulated 45Ca uptake in a similar fashion to that found for cultured rat dorsal root ganglion neurons (DRGs); this response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of C6 cells with capsaicin or RTX produced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the C6 cells corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high affinity [3H]RTX binding site. Consistent with this difference, in C6 cells we were unable to detect [3H]RTX binding. These characteristics suggest the presence of C-type but not R-type vanilloid receptors on C6 cells. After 2 day treatment, capsaicin but not RTX inhibited the proliferation and altered the differentiation of the cells and produced apoptosis. In the long term experiments, capsazepine, instead of antagonizing the effect of capsaicin, acted as an agonist. Moreover, capsazepine displayed these effects with higher potency than that of capsaicin. The different potencies and structure activity relations suggest a distinct mechanism for these long-term vanilloid effects. Our finding that C6 cells can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of response to vanilloids, and highlights the importance of the neuron-glial network., (Copyright 1998 Elsevier Science B.V.)

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. The C-type receptor is coupled to 45Ca uptake, whereas the R-type is detectable by [3H]RTX binding. We describe here specific vanilloid responses in murine mast cells (MCs). In the MC lines and in bone marrow-derived mast cells, capsaicin and RTX induced 45Ca uptake similarly to that observed for cultured rat dorsal root ganglion neurons (DRGs). This response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of MCs with capsaicin or RTX induced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the MCs corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high-affinity [3H]RTX binding site. Consistent with this difference, in MCs we were unable to detect [3H]RTX binding. Vanilloids were noncytotoxic to the MCs, in contrast to the DRGs. Although vanilloids did not cause degranulation in MCs, in the P815 clone capsaicin evoked selective interleukin-4 release. We conclude that certain MCs possess vanilloid receptors, but only the C-type that functions as a channel. Our finding that MCs can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of inflammation in response to vanilloids.

C-fiber sensory afferent neurons, which contain neuropeptides such as calcitonin-gene related peptide and substance P, mediate a wide variety of physiologic responses, including chemogenic pain, thermoregulation, and neurogenic inflammation. Capsaicin, the pungent constituent in red pepper, functions to activate and then, at higher doses and longer times, desensitize this class of neurons. This latter response provides the basis for the therapeutic application of capsaicin. A major advance in the field has been the identification of resiniferatoxin, a phorbol-related diterpene, as an analog of capsaicin that is ultrapotent but with differential selectivity. In particular, resiniferatoxin is only similar in potency for induction of pain but is much more effective for desensitization. Structure-activity analysis in whole animal experiments provides further evidence for dissociation of biologic endpoints, strongly arguing for the existence of vanilloid receptor subclasses. Using resiniferatoxin, we have been able to define specific, high-affinity receptors for capsaicin both in animal models such as rats and in man. Of great importance, the pharmacologic characterization in cultured dorsal root ganglion cells of the high-affinity resiniferatoxin-binding site and of the physiologic response believed to be directly coupled to the receptor, viz. calcium uptake, differed in structure-activity and in cooperativity. We conclude that multiple high-affinity vanilloid receptor subclasses mediate vanilloid response; moreover, the resiniferatoxin-selective subclass of vanilloid receptors is not the voltage-independent, cation-nonselective ion channel as previously believed. Optimization of ligands for the individual vanilloid receptor subclasses should revolutionize this therapeutic area.

Compounds related to capsaicin and its ultrapotent analog, resiniferatoxin (RTX), collectively referred to as vanilloids, interact at a specific membrane recognition site (vanilloid receptor), expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation. Desensitization to vanilloids is a promising therapeutic approach to mitigate neuropathic pain and pathological conditions (e.g. vasomotor rhinitis) in which neuropeptides released from primary sensory neurons play a major role. Capsaicin-containing preparations are already commercially available for these purposes. The use of capsaicin, however, is severely limited by its irritancy, and the synthesis of novel vanilloids with an improved pungency/desensitization ratio is an on-going objective. This review highlights the emerging evidence that the vanilloid receptor is not a single receptor but a family of receptors, and that these receptors recognize not simply RTX and capsaicin structural analogs but are broader in their ligand-binding selectivity. We further focus on ligand-induced messenger plasticity, a recently discovered mechanism underlying the analgesic actions of vanilloids. Lastly, we give a brief overview of the current clinical uses of vanilloids and their future therapeutic potential. The possibility is raised that vanilloid receptor subtype-specific drugs may be synthesized, devoid of the undesirable side-effects of capsaicin.

1. The irritant fungal terpenoid isovelleral caused protective eye-wiping movements in the rat upon intraocular instillation and showed cross-tachyphylaxis with capsaicin, the pungent principle in hot pepper. 2. Isovelleral induced a dose-dependent calcium uptake by rat dorsal root ganglion neurones cultured in vitro with an EC50 of 95 nM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine. 3. Isovelleral inhibited specific binding of [3H]-resiniferatoxin (RTX), an ultrapotent capsaicin analogue, to rat trigeminal ganglion or spinal cord preparations with an IC50 of 5.2 microM; in experiments in which the concentration of [3H]-RTX was varied, isovelleral changed both the apparent affinity (from 16 pM to 37 pM) and the co-operativity index (from 2.1 to 1.5), but not the Bmax. 4. The affinity of isovelleral for inducing calcium uptake or inhibiting RTX binding was in very good agreement with the threshold dose (2.2. nmol) at which it provoked pungency on the human tongue. 5. For a series of 14 terpenoids with an unsaturated 1,4-dialdehyde, a good correlation was found between pungency on the human tongue and affinity for vanilloid receptors on the rat spinal cord. 6. The results suggest that isovelleral-like compounds produce their irritant effect by interacting with vanilloid receptors on capsaicin-sensitive sensory neurones. Since these pungent diterpenes are structurally distinct from the known classes of vanilloids, these data provide new insights into structure-activity relations and may afford new opportunities for the development of drugs targeting capsaicin-sensitive pathways.

Resiniferatoxin (RTX) depletes vanilloid (capsaicin) receptors from lumbar dorsal root ganglia (DRG) of the rat. In addition, RTX causes changes in neuropeptide and nitric oxide synthase expression in lumbar DRG neurons, similar to those described following axotomy; this latter phenomenon is referred to as messenger plasticity. These findings suggested that vanilloid receptor loss may be part of the plasticity that follows RTX treatment. Here we show that vanilloid receptor expression, as detected by [3H]RTX autoradiography, is not changed in lumbar DRGs of axotomized rats, nor is it altered in a rat model (chronic constriction injury) of neuropathic pain. Thus, the in vivo expression of vanilloid receptors detected by specific [3H]RTX binding does not require the presence of intraaxonally transported trophic factors such as nerve growth factor. We conclude that messenger plasticity and vanilloid receptor loss are mediated by distinct mechanisms.

Capsaicin binds to a specific recognition site, referred to as the vanilloid receptor, which it shares with the natural, ultrapotent agonist resiniferatoxin and with the competitive antagonist capsazepine. Upon binding to its receptor, capsaicin opens a cation channel leading to Ca2+ influx. The binding of capsaicin or resiniferatoxin by the vanilloid receptor follows a sigmoidal saturation curve, indicative of positive cooperativity. The biological significance of this positive cooperative behaviour is unknown, as is the mechanism responsible for it. We have developed a novel ligand, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), which binds to cultured rat sensory neurons (with a Ki of 3.1 +/- 0.4 microM), and induces Ca2+ uptake by them (with an ED50 of 1.8 +/- 0.3 microM) with similar affinities and in a non-cooperative manner (Hill coefficients are 0.99 and 1.06 for binding and Ca2+ uptake, respectively). The behaviour of PPAHV thus contrasts with resiniferatoxin or capsaicin not only in the lack of cooperativity but also in the relative potencies for resiniferatoxin binding versus Ca2+ uptake (resiniferatoxin is less potent and capsaicin is more potent for induction of Ca2+ uptake than for binding). In further experiments in which the concentration of [3H]resiniferatoxin was varied, 1 microM PPAHV likewise reduced the cooperativity index that characterizes resiniferatoxin binding to rat spinal cord membranes from 2.3 +/- 0.1 to 1.1 +/- 0.2; in parallel experiments, neither capsaicin nor capsazepine (both at a concentration of 2 microM) affected binding cooperativity. Moreover, PPAHV (1 microM) turned the bi-phasic dissociation curve of resiniferatoxin into a monophasic curve, eliminating the second, slow-dissociation phase. The present results suggest that positive cooperativity is a ligand-induced feature rather than an inherent property of vanilloid receptors. A comparison of the spectrum of biological activity of ligands which bind to vanilloid receptors with different degrees of cooperativity may provide an approach to explore the functional significance of this binding behaviour.

Neuroleptic drugs were reported to modulate [3H]resiniferatoxin binding to vanilloid receptors in the spinal cord, with marked differences between rat and man. In the present study, we have used a [3H]resiniferatoxin binding assay using porcine dorsal horn membranes to explore further species differences in the interaction of neuroleptic drugs at spinal vanilloid receptors. Specific binding of 13 pM [3H]resiniferatoxin to porcine dorsal horn membranes (corresponding to a 7% fractional receptor occupancy) was affected by trifluoperazine in a bi-phasic fashion, with an initial 90% enhancement of binding preceding inhibition: a fit to the modified Hill equation yielded a cooperativity index of 1.8 and a Ki of 5 microM. Under similar conditions, rimcazole, by contrast, had a monophasic effect: it enhanced but, up to 100 microM, did not inhibit [3H]resiniferatoxin binding. These results are in accord with previous findings in human spinal cord but contrast with those in the rat. In experiments in which the concentration of [3H]resiniferatoxin was varied, 20 microM trifluoperazine reduced the Bmax by 33% (from 181 +/- 9 fmol/mg protein to 121 +/- 5 fmol/mg protein) without a measurable change in affinity or cooperativity. In parallel experiments, by contrast, neither capsaicin nor capsazepine (both at a concentration of 10 microM) affected the Bmax or cooperativity but, as expected, reduced the affinity from 61 +/- 8 pM to 120 +/- 11 pM or to 101 +/- 7 pM, respectively. Whereas vanilloid receptor agonists (resiniferatoxin and capsaicin) affected [3H]resiniferatoxin binding at low (approximately 7%) fractional receptor occupancies by the radioligand in a bi-phasic fashion, the competitive vanilloid receptor antagonist capsazepine failed to induce the initial binding enhancement. Thus, capsazepine appears to bind to vanilloid receptors in a non-cooperative fashion, or at least with much reduced positive cooperativity in this system. The mechanism by which neuroleptic drugs modulate resiniferatoxin binding is yet to be clarified and is clearly complicated as well as species-dependent; nonetheless, the reduced Bmax at higher concentrations suggests that it may at least in part be non-competitive.

The [3H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [3H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [3H]RTX bound with similar affinity and positive cooperativity to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 +/- 3.5 pM, 38.4 +/- 3.1 pM and 42.6 +/- 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 +/- 0.11, 1.78 +/- 0.12 and 1.78 +/- 0.09, respectively. The maximal binding capacity was 0.218 +/- 0.026 fmol/10(3) cells and 0.196 +/- 0.021 fmol/10(3) cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [3H]RTX from cells in suspension with Ki and Hill coefficient values of 42.5 +/- 5.3 pM, 2.06 +/- 0.16 microM, 3.16 +/- 0.21 microM and 32.4 +/- 4.1 nM and 1.79 +/- 0.17, 1.68 +/- 0.06, 1.72 +/- 0.11 and 1.81 +/- 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing 45Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of 45Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 +/- 2.9 pM, 25.7 +/- 3.0 nM, 68.5 +/- 3.8 nM, 173 +/- 25 pM, 7.98 +/- 0.83 microM and 4.93 +/- 0.35 microM as compared to 0.94 +/- 0.12 nM, 26.5 +/- 3.5 nM, 149 +/- 30 nM, 1.46 +/- 0.25 nM, 1.41 +/- 0.48 microM and 340 +/- 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperativity of receptor binding; however, stimulation of 45Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [3H]RTX by rat dorsal root ganglion membrane preparations with Ki and Hill coefficient values of 3.89 +/- 0.38 microM and 1.74 +/- 0.11. On the other hand it inhibited the induction of 45Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with Ki values of 271 +/- 29 nM and 325 +/- 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperativity. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of 45Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.

Vanilloid (capsaicin) receptors were visualized by [3H]resiniferatoxin (RTX) autoradiography in the brain of newborn as well as adult (both control and colchicine-treated) rats. Specific labelling was seen in the brain stem only, in the nucleus of the solitary tract extending into the area postrema and the spinal sensory nucleus of the trigeminal nerve. Also, a strong signal was seen in the dorsal horn, dorsal root, trigeminal and nodose ganglia. Membranes obtained from the cervical, thoracic, and lumbar segments of the spinal cord showed similar affinities for RTX and likewise for capsaicin and capsazepine; maximal receptor density was similar in the cervical and thoracic segments (approximately 70 fmol/mg protein) but was twice as high in the lumbar segment. 24 h after ligation of the vagal or the sciatic nerves, a strong accumulation of specific RTX binding sites was observed mainly proximal to the ligature, implying intraaxonal receptor transport from the nodose and dorsal root ganglia, respectively, to the periphery. Systemic (s.c.) vanilloid treatment depleted specific [3H]RTX binding sites from the brain stem, the sensory (dorsal root as well as trigeminal) ganglia, and the spinal cord. RTX was approximately 200-fold more potent than capsaicin for eliminating vanilloid receptors from the spinal cord. The present results suggest a discrete expression of vanilloid receptors in the brain stem (sensory nuclei); although intrinsic vanilloid receptor-expressing neurons are though to exist in the rat brain, they remain undetected by the present [3H]RTX autoradiography methodology.

Optimum treatment of neuropathic pain includes the use of adjuvant analgesics such as antipsychotic drugs and tricyclic antidepressants. Although the mechanism of their analgesic action is not known, it is possible that such agents act directly on pain pathways. The ability of capsaicin and its analogs to selectively deactivate primary afferent neurons provides a basis for their use in human therapy to relieve a number of chronic pain conditions. We examined whether the phenothiazine antipsychotic drug trifluoperazine (TFP) as well as other neuroleptics and tricyclic antidepressants have an effect on the agonist binding properties and the activation of the human and rat vanilloid receptors. Binding of [3H]resiniferatoxin (RTX) to membrane preparations of human dorsal horn and rat whole spinal cord was affected by TFP in a biphasic fashion, with an initial 25 and 65% enhancement of [3H]RTX binding, respectively, preceding inhibition. The apparent Ki values for inhibition were 3.93 +/- 0.13 microM for human dorsal horn and 7.91 +/- 0.62 microM for rat spinal cord. Scatchard analyses revealed that TFP affected both the affinity and the cooperativity of [3H]RTX binding by the receptors, leaving the receptor density unaltered. Similar effects on [3H]RTX binding to rat spinal cord membranes were also induced by other antipsychotic phenothiazines and other types of antipsychotics, by phenothiazines without antipsychotic actions, as well as by tricyclic antidepressants. In cultures of dorsal root ganglion neurones, TFP at concentrations that increased [3H]RTX binding (1-3 microM) also induced an increase in 45Ca uptake; this increase was absent in cultures prepared from capsaicin desensitized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The naturally occurring diterpene resiniferatoxin (RTX) is an ultrapotent analogue of capsaicin. Acting on polymodal afferent neurons, RTX induces a generally similar pattern of responses as does capsaicin. However, the two compounds, as well as other vanilloid derivatives, display different relative potencies for different responses. In the present study, we examined the vanilloid-like activities of two new derivatives, the amide analogue of RTX and phorbol 12,13-dibenzoate 20-homovanillylamide. Structurally, RTX-amide resembles capsaicin more closely than does RTX, and after cleavage of the amide bond the resulting amine would be predicted to not bind to protein kinase C in contrast to resiniferonol 9,13,14-orthophenylacetate, the parent diterpene of RTX. In contrast to our expectations the binding potency of the RTX-amide for the vanilloid receptor present in rat spinal cord was 450-fold lower than that of RTX (Ki values for the RTX-amide and RTX were 10.4 +/- 0.7 nM and 23.1 +/- 3.2 pM, respectively). In the case of phorbol 12,13-dibenzoate 20-homovanillylamide, there was a further loss of affinity for the vanilloid receptor compared with RTX; nonetheless, the Ki (8.56 +/- 0.61 microM) was comparable with that of capsaicin (5.31 +/- 0.37 microM). Computer fitting of the binding data yielded Hill coefficient values of 2.25 +/- 0.03, 2.33 +/- 0.03, and 1.84 +/- 0.05 for RTX, RTX-amide, and phorbol 12,13-dibenzoate 20-homovanillylamide, respectively, indicating that both new compounds induced apparent positive cooperativity among vanilloid binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (Kd = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.

Protons and capsaicin activate overlapping subsets of sensory nerves by opening ion conductances of similar properties. We have used the [3H]resiniferatoxin binding assay utilizing rat spinal cord membranes to elucidate the possible interaction of protons at the vanilloid (capsaicin) receptor. Using low pH (pH 6.0 and pH 5.0) buffers, a time-dependent gradual decrease was observed in specific resiniferatoxin binding. Protons inhibited resiniferatoxin binding with an IC50 of pH 5.3 +/- 0.1. In experiments in which the concentration of [3H]resiniferatoxin was varied, protons reduced the Bmax value by approximately 40% with a corresponding 2-fold decrease in affinity. No change however, was observed in binding cooperativity (the Hill coefficients were 1.7 +/- 0.1 and 1.6 +/- 0.2 in the presence of pH 7.4 and pH 5.0 buffers, respectively). These changes in binding parameters are consistent with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity (Ki = 5.0 +/- 1.0 microM) very similar to that determined in the presence of a pH 7.4 buffer (Ki = 3.0 +/- 1.5 microM). A cyclooxygenase inhibitor, indomethacin (up to 10 microM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I2 and E1, both at 100 microM). We conclude that protons interact at vanilloid receptors in the rat spinal cord; this interaction is either non-competitive or mixed in nature, and probably is not related to prostanoid generation. Protons and/or putative proton-generated mediators might represent endogenous modulators of the vanilloid receptor.

We have measured the dissociation rate of phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, phorbol 12,13-dibutyrate (PDBu), a weak tumor promoter, and 12-deoxyphorbol 13-phenylacetate (dPP), an antitumor promoter, from intact mouse keratinocytes. PDBu and dPP showed a very rapid release from the cells (t1/2 = 1 min), whereas PMA showed a slower release (t1/2 = 9 min). Western blot analysis of the amounts of protein kinase C alpha (PKC alpha) and PKC delta in the soluble fraction and the Triton X-100-soluble particulate fraction revealed that translocation of both isozymes from the soluble to the particulate fraction was reversible when the phorbol esters were washed off. Washes of 5-15 min resulted in complete redistribution of the PKC isozymes when the cells were previously treated with 1 microM dPP or 1 microM PDBu for 5 min. In the case of treatment with 100 or 10 nM PMA, the redistribution required a longer time; nevertheless, the PKC isozymes returned to the soluble fraction within 60 min. Longer initial treatments with PMA, dPP, and PDBu (up to 60 min) translocated PKC in a very similar, completely reversible fashion. We conclude that in this cell line phorbol esters do not induce the conversion of PKC isozymes to an integral membrane state.

n-Chimaerin is a recently described phorbol ester receptor that shares homology in its N-terminal region with the cysteine-rich zinc finger domain of protein kinase C. We have expressed n-chimaerin in insect cells using the baculovirus system and have used the isolated, recombinant n-chimaerin to characterize phorbol ester binding and structure-activity relations, lipid requirements, and inhibitor sensitivity. We find that n-chimaerin expressed in the baculovirus system bound [3H]phorbol 12,13-dibutyrate with high affinity (0.17 +/- 0.01 nM). Although having only a single cysteine-rich zinc finger region compared to two for protein kinase C, n-chimaerin thus closely resembled protein kinase C alpha. n-Chimaerin was likewise virtually indistinguishable from protein kinase C alpha in phorbol ester structure-activity relations, in phospholipid requirements, and in inhibition of binding by sphingosine and calphostin C, protein kinase C inhibitors acting on the regulatory domain. We conclude that a number of typical approaches used to implicate protein kinase C in biological function in cells do not discriminate between the n-chimaerin and protein kinase C classes of phorbol ester receptors.

We report here that we were able to detect the human vanilloid receptor in all three major central endings of primary afferent neurons--in the dorsal horn of the spinal cord, in the cuneate and gracile nuclei and in the spinal nucleus of the trigeminal nerve--and to characterize the binding properties of the receptor in the dorsal horn. Specific [3H]resiniferatoxin (RTX) binding is thought to represent the vanilloid (capsaicin) receptor. [3H]RTX binding to membranes obtained from total human spinal cord and dorsal horn followed sigmoidal saturation kinetics indicating apparent positive cooperativity. The cooperativity index determined by fitting the data to the Hill equation was 1.37 +/- 0.02 in the total spinal cord and 1.77 +/- 0.16 in the dorsal horn. The apparent dissociation constants in whole spinal cord and dorsal horn membranes were 915 +/- 12 and 532 +/- 27 pM; the receptor densities were 140 +/- 6 and 227 +/- 15 fmol/mg protein, respectively. Membrane preparations from the spinal nucleus of the trigeminal nerve and the cuneate and gracile nuclei also bound [3H]RTX in a similar fashion. In parallel experiments, rat spinal cord membranes bound [3H]RTX with 20- to 40-fold higher affinity, somewhat greater positive cooperativity, but at a 3-fold lower receptor density. As predicted by the modified Hill equation, non-radioactive RTX at low receptor occupancy produced biphasic competition curves.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present report we compared the properties of [3H]resiniferatoxin (RTX) binding by the vanilloid receptors present at different parts of the primary afferent neurons of the rat. We found no major differences in either the affinity or the cooperativity of [3H]RTX binding by vanilloid receptors on the cell body, central terminals, peripheral terminals or axons. Specific binding of [3H]RTX to dorsal root ganglia, whole spinal cord, dorsal vagal complex, urinary bladder, and sciatic and vagal nerves all followed sigmoidal saturation kinetics indicating positive cooperativity among the binding sites. The cooperativity indexes determined by fitting the data to the Hill equation were 1.82 +/- 0.11, 2.21 +/- 0.04, 2.55 +/- 0.01, 1.91 +/- 0.11, 2.03 +/- 0.09 and 2.27 +/- 0.04, respectively. The dissociation constants in dorsal root ganglia, spinal cord, dorsal vagal complex, urinary bladder, and sciatic and vagal nerve membranes were 46.5 +/- 2.7, 29.3 +/- 5.1, 28.2 +/- 1.2, 60.8 +/- 4.4, 59.9 +/- 1.9 and 45.2 +/- 0.7 pM; the receptor densities were 219 +/- 14, 48 +/- 5, 67 +/- 1, 32 +/- 7, 61 +/- 9, and 100 +/- 20 fmol/mg protein, respectively. We could not show any major differences in the affinities of capsaicin and capsazepine in inhibition of [3H]RTX binding by the different membrane preparations either. In all cases the initial enhancement of [3H]RTX binding by nonradioactive RTX, capsaicin, and capsazepine confirmed the existence of positive cooperativity among the binding sites. We were unable to detect specific [3H]RTX binding sites in membrane preparations of the preoptic area, locus ceruleus, substantia nigra, striatum and paraventricular nuclei of the rat brain under our present conditions. Our results suggest the uniformity of the vanilloid receptors present at different parts of the primary afferent neuron.

Capsazepine was reported to block capsaicin- and resiniferatoxin (RTX)-induced responses both in vivo and in vitro with Schild plots suggesting a competitive mechanism of action. We have used the [3H]RTX binding assay, thought to represent the vanilloid (capsaicin) receptor, to explore the inhibitory mechanism of capsazepine at the receptor level in the rat. In competition assays, capsazepine inhibited [3H]RTX binding by spinal cord, dorsal root ganglion (DRG) and urinary bladder membranes with similar Ki values of 4.0 +/- 0.3, 3.5 +/- 0.5 and 5.0 +/- 1.0 microM (mean +/- S.E.M.; three determinations), respectively. By contrast, capsazepine was 35- to 50-fold more potent for inhibiting specific [3H]RTX binding in the airways (Ki = 0.12 +/- 0.02 microM; mean +/- S.E.M.; four determinations). In experiments in which the concentration of [3H]RTX was varied, 10 microM capsazepine reduced the affinity of the vanilloid receptor expressed by DRG and spinal cord membranes for [3H]RTX from 15 +/- 3 to 43 +/- 5 pM, and from 20 +/- 3 to 80 +/- 5 pM (mean +/- S.E.M.; three determinations), respectively, without a measurable change in Bmax or in cooperativity index; these shifts in affinity yield Ki values of 5.2 and 3.3 microM for DRG and spinal cord membranes, respectively. Capsaicin inhibited [3H]RTX binding by spinal cord, DRG and urinary bladder membranes with a 6- to 13-fold higher potency than did capsazepine; the Ki values were 0.3 +/- 0.1, 0.6 +/- 0.4 and 0.5 +/- 0.2 microM (mean +/- S.E.M.; three determinations), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific [3H]resiniferatoxin (RTX) binding is thought to represent the postulated vanilloid (capsaicin) receptor. In the present report, this binding has been reevaluated using a modified [3H] RTX binding assay in which the high nonspecific binding, which limited the previous characterization, was reduced by adding alpha 1-acid glycoprotein, a plasma protein that binds RTX, to the usual binding assay after RTX binding by the vanilloid receptor had been terminated. Specific [3H]RTX binding by both dorsal root ganglion (DRG) and spinal cord membranes of the rat followed sigmoidal saturation kinetics indicating apparent positive cooperativity. The cooperativity index determined by fitting the data to the Hill equation was 1.7 in DRG and 1.9 in spinal cord. Apparent dissociation constants were estimated as 24 pM for DRG and 11 pM for spinal cord preparations. As predicted by the modified Hill equation, at low receptor occupancy nonradioactive agonists (RTX, tinyatoxin, capsaicin) produced biphasic competition curves. The initial (enhancement) phase of these curves correlated with the biological potency of the agonist. Dithiothreitol reduced both positive cooperativity and apparent binding affinity; the oxidizing agent 5,5'-dithiobis-(2-nitrobenzoic acid) reduced the cooperativity index without a major effect on binding affinity. These findings suggest that the vanilloid receptor is a receptor cluster in which the subunits cooperate; cooperation is, at least in part, subject to redox modulation.

Using selective surgical ablations we have investigated the localization of vanilloid receptors (specific [3H] resiniferatoxin binding sites) on terminals of the pelvic, hypogastric, and pudendal nerves in the rat urinary bladder. Pelvic and hypogastric nerve resections resulted in 90% and 25% loss of specific [3H] resiniferatoxin (RTX) binding sites, respectively, whilst pudendic nerve resection had no measurable effect on the binding. In control animals, the density of vanilloid receptors was 1.7-fold higher in the neck than in the dome of the urinary bladder; the Bmax values were 57 +/- 8 and 34 +/- 7 fmol/mg protein, respectively. The binding characteristics of the vanilloid receptor were similar in the urinary bladder of the rat and mouse: Kd values were 87 +/- 15 and 61 +/- 11 pM, Bmax values were 37 +/- 2 and 60 +/- 10 fmol/mg protein, respectively. In contrast to the findings for the rat and mouse, in the urinary bladder of the guinea pig and the hamster the low level of specific [3H]RTX binding prevented the detailed characterization of vanilloid receptors. Nonetheless, at a fixed (60pM) concentration of [3H]RTX, specific binding both in the guinea pig and hamster urinary bladder was approximately 20% of that in the rat urinary bladder. In the urinary bladder of newborn rats, as in adults, a single class of specific [3H]RTX binding sites was found which bound RTX with an affinity of 110 +/- 20 pM and with a maximal binding capacity of 30 +/- 5 fmol/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)