Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

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 cooperatively 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/10³ cells and 0.196 ± 0.021 fmol/10³ 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 K_i and Hill coefficient values of 42.5 ± 5.3 pM, 2.06 ± 0.16 μM, 3.16 ± 0.21 μM 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 μM and 4.93 ± 0.35 μM 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 μM and 340 ± 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperatively 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 K_i and Hill coefficient values of 3.89 ± 0.38 μM 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 K_i 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 cooperatively. 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.     

Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.     

3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1-/- mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.     

Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive

The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at approximately 43 degrees C and approximately 53 degrees C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1-10 microM) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1-10 microM) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 microM) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site.     

Effect of Surgical and Chemical Sensory Denervation on Non-neural Expression of the Transient Receptor Potential Vanilloid 1 (TRPV1) Receptors in the Rat

Pretreatment with the ultrapotent capsaicin analog resiniferatoxin (RTX) has been applied as a selective pharmacological tool in inflammation and pain studies to desensitize transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerve endings. The discovery of TRPV1 receptor on non-neural cells challenges systemic RTX desensitization as a method acting exclusively on a population of sensory neurons, but not on non-neural cells. Systemic RTX desensitization was used for chemical denervation and transection of the sciatic and saphenous nerves for surgical denervation in rats. Quantitative real-time PCR and immunohistochemistry were applied to investigate the presence and alterations of the TRPV1 receptor mRNA and protein following chemical and surgical denervation. We provided the first evidence for non-neural TRPV1 immunopositivity and mRNA expression in the rat dorsal paw and plantar skin as well as the oral mucosa. Neither chemical nor surgical denervation influenced the level of TRPV1 receptor mRNA and protein expression in non-neural cells of either skin regions or mucosa. Therefore, RTX and consequently capsaicin remain to be considered as selective neurotoxins for a population of primary afferent neurons.     

Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey: possible site of opiate-CCK analgesic interactions.

125I-Bolton-Hunter sulfated cholecystokinin-8 was used to localize and characterize cholecystokinin (CCK) receptor binding sites in trigeminal and dorsal root ganglia, and in the spinal cord of the rat, rabbit, and monkey. In the rabbit and monkey, a substantial number, 90 +/- 21% and 24 +/- 8%, respectively, of trigeminal and dorsal root ganglion neurons express CCK binding sites. In the spinal cord, the highest concentration of CCK receptors is found in laminae I and II, which is the major termination site of dorsal root ganglia neurons expressing CCK receptor binding sites. Neonatal capsaicin treatment of the rat results in a 70% decline in CCK receptor binding sites in laminae I and II of the spinal cord, indicating that dorsal root ganglia neurons are a major source of CCK receptors in the spinal cord. Pharmacological experiments using selective CCK-A and CCK-B receptor antagonists demonstrate that CCK-B is the prominent CCK receptor subtype in trigeminal and dorsal root ganglia neurons in the rat, rabbit, and monkey. In the rat and rabbit spinal cord, CCK-B binding sites are the prominent subtype, whereas in the monkey cord, CCK-A is the prominent receptor subtype. These results demonstrate that CCK-B receptors are expressed by a substantial percentage of dorsal root ganglion neurons at all spinal levels, and that CCK may antagonize opiate analgesia at the level of the primary afferent neuron itself.     

Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglion membranes

We have previously demonstrated that resiniferatoxin (RTX), an unusual phorbol-related diterpene, induces similar responses in rodents to those induced by capsaicin, the pungent constituent of hot peppers (the genus Capsicum). Strikingly, RTX was 3-4 orders of magnitude more potent than was capsaicin. We report here specific binding of [3H]RTX to particulate preparations from dorsal root ganglia (DRG), a target tissue of both RTX and capsaicin action. The Kd was 0.27 nM for DRG from the rat; the Bmax was 160 fmol/mg. The respective values for pig DRG were 2.2 nM and 730 fmol/mg. Typical phorbol esters did not inhibit [3H]RTX binding. Capsaicin inhibited binding with 10(4)-fold lower affinity than RTX, consistent with the relative in vivo potencies. The specific [3H]RTX binding appears to represent the postulated capsaicin receptor.     

Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.     

Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites

The vanilloid receptor (VR1) protein functions both as a receptor for capsaicin and a transducer of noxious thermal stimuli. To determine the expression and targetting of this protein, we have generated antisera against both the amino and carboxy termini of VR1. Within the dorsal root and trigeminal ganglia of rats, VR1-immunoreactivity (VR1-ir) was restricted to small and medium sized neurons. VR1-ir was transported into both the central and peripheral processes of these primary afferent neurons, as evidenced by: (i) the presence of VR1-ir in nerve fibres and terminals in lamina I and lamina II of the superficial dorsal horn, and the association of VR1-ir with small diameter nerve fibres in the skin and cornea; (ii) the reduction of VR1-ir in the spinal cord after dorsal rhizotomy; and (iii) the accumulation of VR1-ir proximal to sciatic nerve ligation. At the ultrastructural level, VR1-ir was associated with plasma membranes of neuronal perikarya in dorsal root ganglia and nerve terminals in the dorsal horn. VR1-ir was also seen in nerve fibres and terminals in the spinal trigeminal nucleus and nucleus of the solitary tract. Within a large proportion of dorsal root ganglion neurons and the terminals of their axons, VR1-ir was colocalized with staining for the P2X3 purinoceptor, and with binding sites for the lectin IB4. Surprisingly, VR1-ir did not coexist substantially in nerve fibres and terminals that contain substance P and calcitonin gene-related peptide, suggesting complex mechanisms for the release of these neuropeptides in response to capsaicin application.     

Developmental shift of vanilloid receptor 1 (VR1) terminals into deeper regions of the superficial dorsal horn: correlation with a shift from TrkA to Ret expression by dorsal root ganglion neurons

The cloned vanilloid receptor VR1 can be activated by capsaicin and by thermal stimuli. The pattern of nerve terminals that contain VR1 in adult rat spinal cord does not correspond to axons that arise from a single subset of dorsal root ganglion neurons. Thus, we postulated that the basis underlying this complexity might be better understood from a developmental perspective. First, using capsaicin-induced hyperalgesia as a measure of VR1 function, we found that vanilloid receptors were functional as early as postnatal day 10 (P10), although hyperalgesia was of longer duration in adult. Interestingly, the appearance of VR1 protein in terminals of dorsal root ganglion neurons shifts over this postnatal period. From embryonic day 16 to P20, the majority of VR1 protein in the spinal cord was observed in lamina I. As animals matured, VR1 protein became more abundant in lamina II, particularly in the inner portion. Consistent with these observations, the number of dorsal root ganglion neurons coexpressing VR1 and isolectin B4 binding sites doubled while the number of neurons that had both VR1 and substance P remained relatively constant from P2 to P10. In peripheral processes, the number of VR1-positive nerve fibres and terminals in cutaneous structures in postnatal day 10 was half of that in adults. We also show that the association of VR1 with Ret is the reciprocal of the association of VR1 with Trk A. These results suggest that neurotrophins may regulate the extent to which populations of dorsal root ganglion neurons express VR1.     

Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy.

Sensitivity to the pungent vanilloid, capsaicin, defines a subpopulation of primary sensory neurons that are mainly polymodal nociceptors. The recently cloned vanilloid receptor subtype 1 (VR1) is activated by capsaicin and noxious heat. Using combined in situ hybridization and histochemical methods, we have characterized in sensory ganglia the expression of VR1 mRNA. We show that this receptor is almost exclusively expressed by neurofilament-negative small- and medium-sized dorsal root ganglion cells. Within this population, VR1 mRNA is detected at widely varying levels in both the NGF receptor (trkA)-positive, peptide-producing cells that elicit neurogenic inflammation and the functionally less characterized glial cell line-derived neurotrophic factor-responsive cells that bind lectin Griffonia simplicifolia isolectin B4 (IB4). Cells without detectable levels of VR1 mRNA are found in both classes. A subpopulation of the IB4-binding cells that produce somatostatin has relatively low levels of VR1 mRNA. A previously uncharacterized population of very small cells that express the receptor tyrosine kinase (RET) and that do not label for trkA or IB4-binding has the highest relative levels of VR1 mRNA. The majority of small visceral sensory neurons of the nodose ganglion also express VR1 mRNA, in conjunction with the BDNF receptor trkB but not trkA. Axotomy results in the downregulation of VR1 mRNA in dorsal root ganglion cells. Our data emphasize the heterogeneity of VR1 mRNA expression by subclasses of small sensory neurons, and this may result in their differential sensitivity to chemical and noxious heat stimuli. Our results also indicate that peripherally derived trophic factors may regulate levels of VR1 mRNA.     

Anandamide suppression of Na+ currents in rat dorsal root ganglion neurons

Anandamide, the ethanolamide of arachidonic acid, is an endogenous cannabinoid. It is an agonist at CB1 and CB2 cannabinoid receptors as well as the vanilloid receptor, VR1. It is analgesic in inflammatory and neuropathic pain. Both central and peripheral mechanisms are considered to participate in its analgesia. Primary sensory neurons express Na+ currents that are involved in the pathogenesis of pain. We examined the effect of anandamide on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat dorsal root ganglion neurons. Anandamide inhibited both Na+ currents in a concentration-dependent manner. At a membrane potential of -80 mV, the current inhibition was greater in TTX-S than TTX-R currents (K(d); 5.4 microM vs. 38.4 microM). The activation and inactivation became faster in TTX-R current but not in TTX-S current. Anandamide did not alter the activation voltage in either type of current. It, however, produced a hyperpolarizing shift of the steady-state inactivation voltage in both types of currents. The maximum availability at a large negative potential was not reduced by anandamide. Thus, anandamide seems to affect inactivated Na+ channels rather than resting channels. The inhibition of Na+ currents was not reversed by AM 251 (a CB1 antagonist), AM 630 (a CB2 antagonist) or capsazepine (a VR1 antagonist), suggestive of a direct action of anandamide on Na+ channels. The inhibition of Na+ currents in sensory neurons may contribute to the anandamide analgesia.     

Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt-uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration-dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 micromoles) for 10 min followed by a 2-min wash did not produce significant change in the capsaicin-induced labelling. Coapplication of insulin (10 micromoles) with capsaicin, however, potentiated the 50 and 100 nm capsaicin-evoked staining. Insulin itself also produced cobalt labelling in a concentration-dependent manner. The size-frequency distributions of neurons showing capsaicin- or insulin-induced cobalt accumulation were similar. The insulin-induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one-third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation- and/or phosphatidylinositol(4,5)biphosphate hydrolysis-evoked activation of the vanilloid receptor 1. Although the insulin-induced activation of vanilloid receptor 1 seems to be a short-lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.     

Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root anglion membranes

We have previously demonstrated that resiniferatoxin (RTX), an unusual phorbol-related diterpene, induces similar responses in rodents to those induced by capsaicin, the pungent constituent of hot peppers (the genus Capsicum). Strikingly, RTX was 3–4 orders of magnitude more potent than was capsaicin. We report here specific binding of [³H]RTX to particulate preparations from dorsal root ganglia (DRG), a target tissue of both RTX and capsaicin action. The K_d was 0.27 nM for DRG from the rat; the B_max was 160 fmol/mg. The respective values for pig DRG were 2.2 nM and 730 fmol/mg. Typical phorbol esters did not inhibit [³H]RTX binding. Capsaicin inhibited binding with 10⁴-fold lower affinity than RTX, consistent with the relative in vivo potencies. The specific [³H]RTX binding appears to represent the postulated capsaicin receptor.     

Capsaicin inhibits the in vitro binding of peptides selective for mu- and kappa-opioid, and nociceptin-receptors

Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin((1-17)) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1-10 microM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [(3)H]endomorphin-1, [(3)H]endomorphin-2, [(3)H]nociceptin((1-17)) and [(3)H]dynorphin((1-17)). Capsaicin-dependent inhibition was also observed in [(35)S]GTPgammaS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural mu-opioid ligands such as [D-Ala(2),(NMe)Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [(3)H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing mu-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [(3)H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.     

Neurokinin-1 receptor expression in dorsal root ganglion neurons of young rats

The expression of neurokinin-1 receptors was studied in the fourth lumbar dorsal root ganglia of young rats using immunohistochemical and electrophysiological techniques. Use of a specific immunoserum raised against the C-terminal fragment of rat neurokinin-1 receptor revealed immunoreactivity in 32 +/- 1.5% of dorsal root ganglion neurons. The diameter of the majority of the neurokinin-1 receptor immunostained neurons was smaller than 30 microm. Double immunohistochemical labelling using neurokinin-1 receptor and substance P antibodies revealed that about 1/3 of the neurokinin-1 receptor expressing neuron contains substance P. Likewise, about 1/3 of the substance P producing DRG cells expressed the neurokinin-1 receptor. Superfusion of substance P (1 microM) to an in vitro preparation of the fourth lumbar dorsal root ganglion induced a reversible long-lasting depolarization as measured by extracellular suction electrodes attached to the dorsal roots. This response to substance P was only partially antagonized by the selective neurokinin-1 receptor antagonist RP 67580 (1 microM). Intracellular recordings distinguished between Aalpha/beta-, Adelta- and C-sub-types of ganglion neurons. Superfusion of substance P (1 microM) evoked excitatory responses in Adelta- and C-type neurons. These results demonstrate the expression of functional neurokinin-1 receptors on a subpopulation of Adelta- and C-type sensory ganglion neurons. Our data suggest the possible physiological importance of peripheral neurokinin-1 receptors located on dorsal root ganglion neurons.     

Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.     

The putative role of vanilloid receptor-like protein-1 in mediating high threshold noxious heat-sensitivity in rat cultured primary sensory neurons

High threshold noxious heat-activated currents and vanilloid receptor-like protein-1 expression were studied in rat cultured primary sensory neurons to find out the molecule(s) responsible for high threshold noxious heat-sensitivity. The average temperature threshold and amplitude of high threshold noxious heat-activated currents were 51.6 +/- 0.13 degrees C and -2.0 +/- 0.1nA (at a holding potential of -60 mV), respectively. The current-voltage relationship of high threshold noxious heat-activated currents was linear at positive membrane potentials, while it showed a weak inward rectification at negative membrane potentials. The average reversal potential measured in control intracellular and extracellular solutions was 4.5 +/- 0.9 mV (n = 6). Ionic substitutions revealed that the high threshold noxious heat-activated current is a nonselective cationic current with calculated ionic permeabilities of Cs+ : Na+ : Ca2+ (1 : 1.3 : 4.5). Consecutive stimuli reduced the heat threshold from 52.2 +/- 1 to 48.4 +/- 1.4 degrees C and then to 44 +/- 0.7 degrees C (n = 3). High threshold noxious heat-activated currents could dose-dependently and reversibly be reduced by ruthenium red (100 nm-10 micro m) but not by capsazepine (10 micro m). The average longest diameter of high threshold noxious heat-sensitive neurons was 31.48 +/- 0.5 micro m (A = approximately 778 micro m2; n = 77). Twenty-three percent of the total neuronal population expressed vanilloid receptor-like protein-1. The average area of the vanilloid receptor-like protein-1-immunopositive cells was 1,696 +/- 65.3 micro m2 (d = approximately 46 micro m). Vanilloid receptor-like protein-1-expressing neurons did not express the vanilloid receptor 1. Comparison of our data with results obtained in vanilloid receptor-like protein-1-expressing non-neuronal cells and previous immunohistochemical findings suggests that high threshold noxious heat-activated currents are produced by vanilloid receptor-like protein-1 and that high threshold heat-sensitive dorsal root ganglion neurons are the perikarya of type I noxious heat-sensitive fibers.     

Combined determination with functional and morphological studies of origin of nerve fibers expressing transient receptor potential vanilloid 1 in the myenteric plexus of the rat jejunum

The aim of this study was to determine the action of capsaicin in isolated rat intestine and the origin of nerve fibers expressing transient receptor potential vanilloid 1 (TRPV1: capsaicin receptor) in the rat jejunum by combination of functional and immunohistochemical experiments. Capsaicin (1 microM) produced a prolonged relaxation response (52. +/-15.3% of the relaxation response to papaverine, mean +/- S.D., n=27) of the isolated jejunum in the presence of atropine and guanethidine. Pretreatment with the TRPV1 antagonist, capsazepine (10 microM) and ruthenium red (3 microM) significantly reduced the relaxation response to capsaicin by 78% (P<0.01) and 38% (P<0.05), respectively. Tetrodotoxin and calcitonin gene-related peptide (CGRP)-desensitization significantly reduced the response to capsaicin by 72% (P<0.01) and 42% (P<0.01), respectively. Therefore, we investigated the distribution of TRPV1-immunoreactivity (IR) in the myenteric plexus of the rat jejunum. Using antisera raised against either the N-terminal or C-terminal domains of rat TRPV1, TRPV1-IR was present in the nerve fibers, but not in the cell bodies of myenteric neurons. These TRPV1-immunoreactive nerve fibers were running in myenteric ganglia and their interconnecting strands. Most TRPV1-immunoreactive nerve fibers showed CGRP-IR, whereas few VR1-immunoreactive nerve fibers showed substance P-IR. After chronic denervation of the extrinsic nerve supply to the jejunum, both the relaxation response to capsaicin and TRPV1-immunoreactive nerve fibers completely disappeared. These findings indicate that these TRPV1-immunoreactive nerve fibers in the rat jejunum derive from extrinsic neurons and that activation of TRPV1 produces the relaxation response in the rat jejunum, at least in part, through the release of CGRP from nerve fibers expressing TRPV1.     

Vanilloid receptor TRPV1 is not activated by vanilloids applied intracellularly

The vanilloid receptor TRPV1 is a ligand-gated cation channel that can be activated by capsaicin, acids and noxious heat. For vanilloids, a stretch of approximately 8 amino acids in the vicinity of the TM3 region on the cytosolic side of TRPV1 and R114 and E761 in the N- and C-cytosolic tails, respectively, has been shown to be critical for capsaicin binding and channel activation. Here, we report that intracellular application of vanilloids is insufficient for activating TRPV1 channels in HEK293T cells. Pipette solution (ICS) for recording membrane currents was supplemented with 50 microM capsaicin (n=14) or 1 microM resiniferatoxin (RTX) (n=39) and the responses induced by extracellular capsaicin (1 microM) or RTX (100 nM) were recorded at intervals >50% of that needed for diffusion of Lucifer yellow from the pipette to reach maximum fluorescence (n=7). We found that all cells with expressed TRPV1 exhibited a similar sensitivity to vanilloids irrespective of whether the membrane currents were recorded with electrodes filled with ICS containing capsaicin or RTX or only with control ICS. We suggest that, in addition to intracellularly located agonist recognition sites of TRPV1, there is at least one resides on the extracellular side, which needs to be occupied to activate the channel.