Receptors In Rat Spinal Cord Research Articles

Electroacupuncture at Hua Tuo Jia Ji Acupoints Reduced Neuropathic Pain and Increased GABAA Receptors in Rat Spinal Cord.

Chronic constriction injury- (CCI-) induced neuropathic pain is the most similar model to hyperalgesia in clinical observation. Neuropathic pain is a neuronal dysfunction in the somatosensory system that may lead to spontaneous pain. In this study, electroacupuncture (EA) was applied at bilateral L4 and L6 of Hua Tuo Jia Ji points (EX-B2) for relieving neuropathic pain in rats. Eighteen Sprague-Dawley rats were randomly assigned to three groups: sham, 2-Hz EA, and 15-Hz EA groups. Following von Frey and cold plate tests, both the 2- and the 15-Hz EA groups had significantly lower mechanical and thermal hyperalgesia than the sham group. Western blot analysis results showed that γ-aminobutyric acid A (GABAA), adenosine A1 receptor (A1R), transient receptor potential cation channel subfamily V member 1 (TRPV1), TRPV4, and metabotropic glutamate receptor 3 (mGluR3) were similar in the dorsal root ganglion of all three groups. Furthermore, levels of GABAA receptors were higher in the spinal cord of rats in the 2- and 15-Hz EA groups compared with the sham control group. This was not observed for A1R, TRPV1, TRPV4, or mGluR3 receptors. In addition, all the aforementioned receptors were unchanged in the somatosensory cortex of the study rats, suggesting a central spinal effect. The study results provide evidence to support the clinical use of EA for specifically alleviating neuropathic pain.

Simultaneous inhibition of NMDA and mGlu1/5 receptors by levo‐corydalmine in rat spinal cord attenuates bone cancer pain

Bone cancer pain is a challenge for its not completely clarified mechanism and broad clinical morbidity. Therefore, novel and more effective drugs are urgent needed for improvement of patients' quality of life. Glutamate receptors have been associated with the development of the central sensitization of chronic pain. Inhibition of N-methyl-d-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors can effectively attenuate bone cancer pain, respectively. Herein, our results indicated that levo-Corydalmine (l-CDL), a compound from Corydalis yanhusuo W.T. Wang, which has been used in traditional Chinese medicine for pain relief could effectively attenuate bone cancer pain induced by tibia bone cavity tumor cell implantation (TCI) through simultaneously inhibiting the NMDA and mGlu1/5 receptors in rat spinal cord without notable side effects. Both intragastric and intrathecal administration of l-CDL significantly alleviated the mechanical hypersensitivity induced by TCI in rats, and the analgesic effect of l-CDL could be reversed by intrathecal administration of NMDA receptor agonist NMDA and mGlu1/5 receptor agonist DHPG but not AMPA receptor agonist AMPA. l-CDL could also selectively suppress NMDA and DHPG induced rapid rise in Ca2+ oscillations in primary cultures neurons of spinal cord in vitro. The antinociception of l-CDL were partially mediated by the reduced phosphorylation of PKC γ and ERK1/2 in spinal cord of TCI rats in a NMDA and mGlu1/5 dependent manner. In conclusion, these results suggested that l-CDL attenuates TCI induced bone cancer pain through simultaneously inhibiting the NMDA and mGlu1/5 receptors and the downstream PKC γ, ERK1/2 signaling pathways in the spinal cord.

Intrathecal orexin A increases sympathetic outflow and respiratory drive, enhances baroreflex sensitivity and blocks the somato‐sympathetic reflex

Intrathecal (i.t.) injection of orexin A (OX-A) increases blood pressure and heart rate (HR), but the effects of OX-A on sympathetic and phrenic, nerve activity, and the baroreflex(es), somato-sympathetic and hypoxic chemoreflex(es) are unknown. Urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats were examined in this study. The effects of i.t. OX-A (20 nmol 10 µL⁻¹) on cardiorespiratory parameters, and responses to stimulation of the sciatic nerve (electrical), arterial baroreceptors (phenylephrine hydrochloride, 0.01 mg kg⁻¹ i.v.) and peripheral (hypoxia) chemoreceptors were also investigated. i.t. OX-A caused a prolonged dose-dependent sympathoexcitation, pressor response and tachycardia. The peak effect was observed at 20 nmol with increases in mean arterial pressure, HR and splanchnic sympathetic nerve activity (sSNA) of 32 mmHg, 52 beats per minute and 100% from baseline respectively. OX-A also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude and a fall in phrenic nerve frequency, an increase in neural minute ventilation, a lengthening of the expiratory period, and a shortening of the inspiratory period. All effects of OX-A (20 nmol) were attenuated by the orexin receptor 1 antagonist SB 334867. OX-A significantly reduced both sympathoexcitatory peaks of somato-sympathetic reflex while increasing baroreflex sensitivity. OX-A increased the amplitude of the pressor response and markedly amplified the effect of hypoxia on sSNA. Thus, activation of OX receptors in rat spinal cord alters cardiorespiratory function and differentially modulates sympathetic reflexes.

Studies on expression of FSH and its anti-apoptotic effects on ischemia injury in rat spinal cord

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia-reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.

Effects of acute agonist treatment on subcellular distribution of κ opioid receptor in rat spinal cord

We investigated whether acute treatment with agonists affected the subcellular distribution of kappa opioid receptor (KOPR) in the dorsal horn of the rat lumbar spinal cord by using immunoelectron microscopy. Rats were injected intrathecally (i.t.) with U50,488H (100 nmole), dynorphin A(1-17) (15 nmole), or vehicle. The doses chosen have been shown to induce antinociception. Rats were perfused transcardially 30 min later, and lumbar spinal cords were removed and processed for electron microscopic analysis. KOPR was stained with KT-2, a specific polyclonal antibody against the rat/mouse KOPR(371-380) peptide, followed by gold-labeled secondary antibody and silver intensification. The silver grains were present in axons, terminals, dendrites, and somata, and the association with plasma membranes was quantified in dendrites, because KOPR immunoreactivity was most frequently observed in these profiles. In vehicle-treated rats, approximately 27% of KOPR immunoreactivity was associated with plasma membranes. U50,488H, i.t., did not cause a significant change in the percentage of KOPR present on plasma membranes, whereas dynorphin A, i.t., significantly decreased cell surface KOPR to approximately 19%. In summary, these data indicate that U50,488H and dynorphin A differentially regulate the subcellular distribution of endogenous KOPR.

Biochemical anti-opioid action of NPFF 2 receptors in rat spinal cord

Neuropeptide FF (NPFF) agonists counteract the cellular opioid actions. We demonstrate for the first time a biochemical anti-opioid effect of NPFF receptors in the rat spinal cord by using the [ 35S]GTPγS binding assay in autoradiography. The mu agonist DAMGO as well as the potent and selective NPFF 2 agonist dNPA, stimulated [ 35S]GTPγS binding at different optimal GDP concentrations. dNPA decreased the effects induced by DAMGO alone; the maximal of G-protein coupling was decreased but not the potency of opioid agonist. We conclude that NPFF 2 receptors are coupled to G-protein in the rat spinal cord and could exert a molecular anti-opioid effect.

Effect of remifentanil on NMDA receptors in rat spinal cord: an electrophysiological study

Effect of remifentanil on NMDA receptors in rat spinal cord: an electrophysiological study

Protein phosphatase regulates the phosphorylation of NR2B subunit of NMDA receptors in rat spinal cord following intradermal injection of capsaicin

Central sensitization is sensory hypersensitivity in the central nervous system that can be induced by peripheral noxious stimulation. It can be mediated through the activation of several second messenger cascades, such as protein kinase A or protein kinase C. An opposing enzyme to protein kinases, serine/threonine protein phosphatase 2A (PP2A), which catalyzes dephosphorylation reactions, plays an important role in central sensitization. During central sensitization the function of the NMDA receptor is enhanced by increases in channel opening due to its phosphorylation. The effect of a specific inhibitor of PP2A, fostriecin, on the expression of NR2B and phospho-NR2B subunits of NMDA receptors in the spinal cord of rats following capsaicin injection were investigated in this study. After continually perfusing with ACSF or fostriecin (3 M) through a previously implanted microdialysis fiber for 30 minutes, central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw of rats. The spinal cord was removed quickly at different time points (30, 60, 90, 120, 180 min) after intradermal injection of capsaicin. Western blots were performed to examine the expression of NR2B and phospho-NR2B subunits in spinal cord tissue by using specific antibodies. Both NR2B and phospho-NR2B expression were found in ACSFand fostriecintreated groups. At each time point after capsaicin injection, no significant difference was found in NR2B expression in either group. In the ACSF group, phospho-NR2B expression was increased at a time point of 30 minutes and the expression gradually decreased in a time dependent manner. In fostriecin group, phosphoNR2B expression was not significantly reduced until 180 minutes after capsaicin injection. These results suggest that PP2A has a regulatory effect on central sensitization induced by noxious stimuli in the periphery by modulating the phosphorylation state of NMDA receptors. Supported by NIH grant NS09743.

Repeated Restraint Stress Reduces Opioid Receptor Binding in Different Rat CNS Structures

Different effects of exposure to acute or to repeated stress have been observed upon the nociceptive response in rats. In the present study, we repeatedly submitted Wistar rats to restraint for 40 days, a treatment known to induce an increase in the nociceptive response in the tail-flick test. Afterwards, the effect of repeated restraint stress on the density of opioid receptors in rat spinal cord, frontal cortex, and hippocampus was investigated. Results showed that repeatedly stressed rats displayed a significant decrease in opioid receptors density in all structures studied; cortex (141.3 +/- 5.7 for control and 103.3 +/- 15.9 for stressed rats), hippocampus (92.4 +/- 7.2 for control and 64.8 +/- 7.7 for stressed rats), and spinal cord (122.2 +/- 12.8 for control and 79.7 +/- 9.7 for stressed rats). These findings suggest opioid mediation of the altered responses observed in these repeatedly-stressed animals, although the participation of non-opioid mechanisms in this phenomenon cannot be ruled out.

Kappa opioid receptors in rat spinal cord: sex-linked distribution differences

Activation of κ opioid receptors (KORs) in the spinal cord can diminish nociception. Humans and rodents show sex differences in the analgesia produced by KOR agonists, and female rats show fluctuations in KOR density and sensitivity across the estrous cycle. However, it is unclear whether there are sex differences in the amount and/or distribution of spinal KORs. In the present study, immunocytochemically labeled KORs were examined in laminae I and II of the lumbosacral spinal dorsal horn of male and normally cycling female Sprague–Dawley rats. The basic pattern of KOR labeling was determined in both sexes using qualitative electron microscopy (EM), and sex-linked differences in the density and subcellular distribution of KOR immunoreactivity were determined with quantitative EM and light microscopy. KOR labeling was visualized with immunoperoxidase for optimally sensitive detection, or with immunogold for precise subcellular localization. By EM, the general pattern of KOR immunoreactivity was similar in males and females. KOR immunoreactivity was common in dendrites, axons, and axon terminals, and was in a few glia and neuronal somata. Most KOR-immunoreactive (-ir) axons were fine-diameter and unmyelinated. Most KOR-ir terminals were small or medium-sized, and a minority formed asymmetric or symmetric synapses with unlabeled dendrites. KOR immunoreactivity was associated both with the plasma membrane and with cytoplasmic organelles, notably including dense core vesicles in terminals. Light microscopic densitometry revealed that KOR immunoreactivity was significantly denser in estrus and proestrus females than in males. By EM, the distribution of KOR-immunogold labeling within axon terminals differed, with a greater proportion of cytoplasmic KOR labeling in estrus females compared with males. In contrast, the abundance and types of KOR-immunoperoxidase-labeled profiles did not show sex-linked differences. We conclude that in both sexes, KORs are positioned to influence both pre- and postsynaptic neurotransmission and are present in morphologically heterogeneous neuron populations. These findings are consistent with complex consequences of KOR activation in the spinal cord. In addition, the presence of increased KOR density and proportionally elevated intracellular KORs in proestrus/estrus females suggests a basis for sex-linked differences in KOR-mediated antinociception.

Antinociceptive role of 5-HT1A receptors in rat spinal cord

Intrathecal administration of 5-hydroxytryptamine (5-HT) is antinociceptive to noxious heat and electrical stimuli. The contributions of different receptor subtypes to the antinociceptive effects of 5-HT are controversial. The main reasons for this are the poor receptor subtype selectivity of some agonist drugs and the difficulty of restricting drug action to the spinal cord in some experimental paradigms. This study investigated the roles of different 5-HT receptor subtypes involved in the spinal cord control of the nociception produced by these two nociceptive testing paradigms. Tail-flick latency and electric current threshold for nociception were measured in an acute pain model that allowed the study of the antinociceptive effects of intrathecally administered drugs that were due to actions of these drugs at spinal cord receptors. Experiments were performed in male Wistar rats with chronically implanted lumbar subarachnoid catheters. Dose-response curves for spinally mediated antinociceptive effects of agonists selective for 5-HT receptor subtypes were constructed. The 5-HT1 agonist 1-(3-chlorophenyl)-piperazine dihydrochloride caused a dose-dependent antinociceptive effect, measured by both nociceptive tests. However, 8-hydroxy-DPAT (selective 5-HT1A agonist) produced antinociception assessed by electric current but not tail flick. A 5-HT1A-selective antagonist, 4-[3-(benzotriazol-1-yl)propyl]-1-(2-methoxyphenyl)-piperazine, reversed the antinociception in the electrical test produced by both of these agonists but the tail-flick latency effects after intrathecal 1-(3-chlorophenyl)-piperazine were not suppressed by this antagonist. We conclude that 5-HT1A receptors in the spinal cord are involved in the nociceptive mechanisms assessed by noxious electrical stimuli. Other 5-HT1 receptors (non 5-HT1A receptors) are involved in the spinally mediated antinociception assessed by thermal noxious stimuli.

D 3 dopamine receptors in rat spinal cord: implications for sensory and motor function

Quantitative autoradiography was used to determine the distribution of D 3 receptors in rat spinal cord and compare it with the distribution of D 1-like and D 2 (and D 4) receptors. [ 3H]PD 128907-labeled D 3 sites were observed in roughly 6-fold lower density than [ 3H]spiperone-labeled D 2 (D 4) sites and 60-fold lower density than [ 3H]SCH 23390-labeled D 1-like sites. Highest densities of D 3 binding were observed in the superficial layers of the dorsal horn at cervical and lumbar levels followed by the pars centralis and dorsal horn. Lowest densities of D 3 sites were detected in the ventral horn. These observations suggest that spinal D 3 receptors may play a role in sensory and/or motor function or contribute to the pharmacological effects of dopaminergic drugs.

Co-localization of N-methyl- d-aspartate receptors and substance P (neurokinin-1) receptors in rat spinal cord

Glutamate, substance P (SP), and their receptors have been implicated in the initiation and maintenance of persistent pain through an interaction at second order spinal cord neurons. Employing well-characterized antibodies to the SP receptor and the N-methyl- d-aspartate receptor (NR1 subunit, splice variant missing exon 22), we demonstrate co-localization of these receptors on second order neurons at cervical, thoracic, lumbar, and sacral spinal cord levels. The co-localization was marked in lamina I of the dorsal horn at all levels and in the intermediolateral nucleus of the thoraco-lumbar spinal cord nuclei associated with autonomic function.

Kappa opioid receptors in rat spinal cord vary across the estrous cycle

Kappa opioid receptors (KORs) were immunocytochemically localized in the lumbosacral spinal cord of female rats in different stages of the estrous cycle to examine the influence of hormonal status on receptor density. KOR labeling was primarily in fine processes and a few neuronal cell bodies in the superficial dorsal horn and the dorsolateral funiculus. Quantitative light microscopic densitometry of the superficial dorsal horn revealed that rats in diestrus had significantly lower KOR densities than those in proestrus or estrus. This suggests that female reproductive hormones regulate spinal KOR levels, which may contribute to variations in analgesic effectiveness of KOR agonists across the estrous cycle.

Pre- and postsynaptic distribution of cannabinoid and mu opioid receptors in rat spinal cord

In vitro receptor binding and quantitative autoradiography were used to assess the pre- and postsynaptic distribution of cannabinoid receptors in the cervical dorsal horn of the rat spinal cord. An extensive unilateral dorsal rhizotomy was performed across seven or eight successive spinal segments from C3 to T1 or T2. The densities of cannabinoid and mu opioid receptors in the central (C6) spinal segment were assessed 2, 4, 8, and 16 days post rhizotomy and compared with those of untreated rats. Rhizotomy induced approximately a 50% ipsilateral loss in the [ 3 H ]CP55,940 binding to spinal cannabinoid receptors that was maximal at 8 days post-rhizotomy. By comparison, the binding of [ 3 H ][ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin (DAMGO) to mu receptors was depleted approximately 60% in near-adjacent sections. By contrast, changes in [ 3 H ]CP55,940 binding contralateral to the deafferentation were largely absent at all post-lesion delays. These data suggest that under conditions in which a spinal segment is completely deafferented, approximately 50% of cannabinoid receptors in the cervical (C6) dorsal horn reside presynaptically on central terminals of primary afferents. The present data provide anatomical evidence for presynaptic as well as postsynaptic localization of cannabinoid receptors in the spinal dorsal horn.

Cuneiform nucleus stimulation-induced sympathoexcitation: role of adrenoceptors, excitatory amino acid and serotonin receptors in rat spinal cord

Stimulation of the midbrain cuneiform nucleus has previously been shown to produce increases in arterial blood pressure and lumbar sympathetic nerve activity. While this sympathoexcitatory effect is, in part, due to excitation of premotor sympathoexcitatory neurons in the rostral ventrolateral medulla, the specific spinal neurotransmitter systems recruited by cuneiform nucleus stimulation remains to be elucidated. In this study, mean arterial pressure, resting and cuneiform nucleus stimulation-evoked lumbar sympathetic nerve activity were analysed following intrathecal injections of an excitatory amino acid antagonist (kynurenic acid), α 1-adrenoceptor antagonist (prazosin) and a serotonin receptor antagonist (methiothepin) in anesthetized, paralysed male Sprague-Dawley rats. Mean arterial pressure and resting sympathetic nerve discharge were decreased by all treatments ( n=6/group) compared to the vehicle control group. Intermittent electrical stimulation of the cuneiform nucleus produced a bimodal sympathoexcitatory response, of which the short latency peak was significantly attenuated (43% reduction) by intrathecal kynurenate whereas the long latency peak was reduced by intrathecal prazosin (decrease of 21%) and methiothepin (38% attenuation). These results are consistent with the significant roles of excitatory amino acid, α 1-adrenergic and serotonin receptors in modulating the activity of sympathetic vasomotor preganglionic neurons supplying the lumbar sympathetic nerve trunk, and suggest the existence of at least three neuronal groups and/or pathways associated with the sympathoexcitatory response to cuneiform nucleus stimulation.

Antinociceptive action of intrathecally administered IGF-I and the expression of its receptor in rat spinal cord

mRNA transcripts for insulin-like growth factor I (IGF-I) and its receptor are expressed in the lumbar region of the spinal cord. Accordingly, we examined the involvement of IGF-I in nociceptive transmission. An intrathecal injection of IGF-I (200–1000 ng) produced a dose-dependent elevation in nociceptive threshold as indicated by tail flick/withdrawal latency. In contrast, comparable doses of insulin had no significant effect. The time-response curve (15–75 min) revealed that the peak for IGF-I's antinociceptive effect is attained at 30 min. Our data provide evidence that the IGF-I system within the spinal cord may serve as a target for novel analgesics.

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization.

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.

New high affinity peptide antagonists to the spinal galanin receptor

1. The role of endogenous galanin in somatosensory processing has been studied with galanin receptor antagonists. The new galanin receptor ligands C7, M32, M38 and M40 bind with high affinity (Kd in nanomolar range) to spinal cord galanin receptors and possess oxidative stability as compared to earlier generations of peptide ligands. These peptides have been examined in the spinal flexor reflex model where exogenous galanin exhibited biphasic excitatory and inhibitory effects. 2. Intrathecal administration of C7 [galanin(1-13)-spantide] and M32 [galanin (1-13)-neuropeptide Y(25-36) amide] blocked facilitation of the nociceptive flexor reflex induced by 30 pmol intrathecal galanin in decerebrate, spinalized rats in a dose-dependent manner, thus behaving as antagonists of the galanin receptor. In contrast, M38[galanin(1-13)-(Ala-Leu)3-Ala amide] and M40 [galanin(1-13)-Pro-Pro-(Ala-Leu)2-Ala amide], exhibited only weak antagonism at high doses in this model. Moreover, lower doses of M40 potentiated galanin-induced reflex facilitation. C7 was neurotoxic at high doses in the rat spinal cord. 3. M32 and C7 were potent antagonists of galanin receptors in rat spinal cord, in correlation with their in vitro binding characteristics. In contrast, M38 and M40, despite their high in vitro affinity, exhibited only very weak antagonism. Moreover, M40 may also behave as a partial agonist. 4. Previous studies have shown that the galanin receptor may be heterogeneous. The discrepancy between in vitro binding and in vivo antagonistic potency of M38 and M40 may also suggest the presence of different galanin receptor subtypes within the rat spinal cord. However, other explanations for the discrepancy, such as differences in metabolic stability, diffusion rates and penetration to the site of action are also possible.

Proton inhibition of [ 3H]resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord

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 IC 50 of pH 5.3 ± 0.1. In experiments in which the concentration of [ 3H]resiniferatoxin was varied, protons reduced the max 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 consist with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity ( K i=5.0 ± 1.0 μM) very similar to that determined in the presence of a pH 7.4 buffer ( K i = 3.0 ± 1.5 μM). A cyclooxygenase inhibitor, indomethacin (up to 10 μM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I 2 and E 1, both at 100 μM). 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.