Iodoresiniferatoxin Research Articles

Analgesic effects of FAAH inhibitor in the insular cortex of nerve-injured rats.

The insular cortex is an important region of brain involved in the processing of pain and emotion. Recent studies indicate that lesions in the insular cortex induce pain asymbolia and reverse neuropathic pain. Endogenous cannabinoids (endocannabinoids), which have been shown to attenuate pain, are simultaneously degraded by fatty acid amide hydrolase (FAAH) that halts the mechanisms of action. Selective inhibitor URB597 suppresses FAAH activity by conserving endocannabinoids, which reduces pain. The present study examined the analgesic effects of URB597 treatment in the insular cortex of an animal model of neuropathic pain. Under pentobarbital anesthesia, male Sprague–Dawley rats were subjected to nerve injury and cannula implantation. On postoperative day 14, rodents received microinjection of URB597 into the insular cortex. In order to verify the analgesic mechanisms of URB597, cannabinoid 1 receptor (CB1R) antagonist AM251, peroxisome proliferator-activated receptor alpha (PPAR alpha) antagonist GW6471, and transient receptor potential vanilloid 1 (TRPV1) antagonist Iodoresiniferatoxin (I-RTX) were microinjected 15 min prior to URB597 injection. Changes in mechanical allodynia were measured using the von-Frey test. Expressions of CB1R, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), and TRPV1 significantly increased in the neuropathic pain group compared to the sham-operated control group. Mechanical threshold and expression of NAPE-PLD significantly increased in groups treated with 2 nM and 4 nM URB597 compared with the vehicle-injected group. Blockages of CB1R and PPAR alpha diminished the analgesic effects of URB597. Inhibition of TRPV1 did not effectively reduce the effects of URB597 but attenuated expression of NAPE-PLD compared with the URB597-injected group. In addition, optical imaging demonstrated that neuronal activity of the insular cortex was reduced following URB597 treatment. Our results suggest that microinjection of FAAH inhibitor into the insular cortex causes analgesic effects by decreasing neural excitability and increasing signals related to the endogenous cannabinoid pathway in the insular cortex.

N-palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor-mediated mechanism.

The neural substrates and mechanisms mediating the antinociceptive effects of the endogenous bioactive lipid, N-palmitoylethanolamide (PEA), require further investigation. We investigated the effects of exogenous PEA administration into the anterior cingulate cortex (ACC), an important brain region linked with cognitive and affective modulation of pain, on formalin-evoked nociceptive behaviour in rats. Potential involvement of peroxisome proliferator-activated receptor isoforms (PPAR) α and γ or endocannabinoid-mediated entourage effects at cannabinoid1 (CB1) receptors or transient receptor potential subfamily V member 1 (TRPV1) in mediating the effects of PEA was also investigated. Intra-ACC administration of PEA significantly attenuated the first and early second phases of formalin-evoked nociceptive behaviour. This effect was attenuated by the CB1 receptor antagonist AM251, but not by the PPARα antagonist GW6471, the PPARγ antagonist GW9662, or the TRPV1 antagonist 5'-iodo resiniferatoxin. All antagonists, administered alone, significantly reduced formalin-evoked nociceptive behaviour, suggesting facilitatory/permissive roles for these receptors in the ACC in inflammatory pain. Post-mortem tissue analysis revealed a strong trend for increased levels of the endocannabinoid anandamide in the ACC of rats that received intra-ACC PEA. Expression of c-Fos, a marker of neuronal activity, was significantly reduced in the basolateral nucleus of the amygdala, but not in the central nucleus of the amygdala, the rostral ventromedial medulla or the dorsal horn of the spinal cord. In conclusion, these data indicate that PEA in the ACC can reduce inflammatory pain-related behaviour, possibly via AEA-induced activation of CB1 receptors and associated modulation of neuronal activity in the basolateral amygdala.

Involvement of apoptosis and calcium accumulation through TRPV1 channels in neurobiology of epilepsy

Calcium ion accumulation into the cytosol of the hippocampus and dorsal root ganglion (DRG) are main reasons in etiology of epilepsy. Transient receptor potential vanilloid type 1 (TRPV1) channel is a cation-permeable calcium channel found in the DRG and hippocampus. Although previous studies implicate TRPV1 channels in the generation of epilepsy, suppression of ongoing seizures by TRPV1 antagonists has not yet been investigated. We tested the effects of TRPV1-specific antagonists, capsazepine (CPZ) and 5′-iodoresiniferatoxin (IRTX) on the modulation of calcium accumulation, apoptosis and anticonvulsant properties in the hippocampus and DRG of pentylentetrazol (PTZ) and capsaicin (CAP) administrated rats.Forty rats were divided into five groups as follows; control, PTZ, CAP+PTZ, IRTX, and IRTX+PTZ. Fura-2 and patch-clamp experiments were performed on neurons dissected from treated animals by CAP and CPZ. PTZ and CAP+PTZ administrations increased intracellular free Ca2+ concentrations, TRPV1 current densities, apoptosis, caspase 3 and 9 values although the values were reduced by IRTX and CPZ treatments. Latency time was extended by application CPZ and IRTX although CAP produced acceleration of epileptic seizures.Taken together, these results support a role for TRPV1 channels in the inhibition of apoptosis, epileptic seizures and calcium accumulation, indicating that TRPV1 inhibition may possibly be a novel target in the DRG and hippocampus for prevention of epileptic seizures and peripheral pain.

TRPV1 Channel: A Potential Drug Target for Treating Epilepsy.

Epilepsy has 2-3% incidence worldwide. However, present antiepileptic drugs provide only partial control of seizures. Calcium ion accumulation in hippocampal neurons has long been known as a major contributor to the etiology of epilepsy. TRPV1 is a calcium-permeable channel and mediator of epilepsy in the hippocampus. TRPV1 is expressed in epileptic brain areas such as CA1 area and dentate gyrus of the hippocampus. Here the author reviews the patent literature on novel molecules targeting TRPV1 that are currently being investigated in the laboratory and are candidates for future clinical evaluation in the management of epilepsy. A limited number of recent reports have implicated TRPV1 in the induction or treatment of epilepsy suggesting that this may be new area for potential drugs targeting this debilitating disease. Thus activation of TRPV1 by oxidative stress, resiniferatoxin, cannabinoid receptor (CB1) activators (i.e. anandamide) or capsaicin induced epileptic effects, and these effects could be reduced by appropriate inhibitors, including capsazepine (CPZ), 5'-iodoresiniferatoxin (IRTX), resolvins, and CB1 antagonists. It has been also reported that CPZ and IRTX reduced spontaneous excitatory synaptic transmission through modulation of glutaminergic systems and desensitization of TRPV1 channels in the hippocampus of rats. Immunocytochemical studies indicated that TRPV1 channel expression increased in the hippocampus of mice and patients with temporal lobe epilepsyTaken together, findings in the current literature support a role for calcium ion accumulation through TRPV1 channels in the etiology of epileptic seizures, indicating that inhibition of TRPV1 in the hippocampus may possibly be a novel target for prevention of epileptic seizures.

Activation of the TRPV1 cation channel contributes to stress-induced astrocyte migration.

Astrocytes provide metabolic, structural, and synaptic support to neurons in normal physiology and also contribute widely to pathogenic processes in response to stress or injury. Reactive astrocytes can undergo cytoskeletal reorganization and increase migration through changes in intracellular Ca(2+) mediated by a variety of potential modulators. Here we tested whether migration of isolated retinal astrocytes following mechanical injury (scratch wound) involves the transient receptor potential vanilloid-1 channel (TRPV1), which contributes to Ca(2+)-mediated cytoskeletal rearrangement and migration in other systems. Application of the TRPV1-specific antagonists, capsazepine (CPZ) or 5'-iodoresiniferatoxin (IRTX), slowed migration by as much as 44%, depending on concentration. In contrast, treatment with the TRPV1-specific agonists, capsaicin (CAP) or resiniferatoxin (RTX) produced only a slight acceleration over a range of concentrations. Chelation of extracellular Ca(2+) with EGTA (1 mM) slowed astrocyte migration by 35%. Ratiometric imaging indicated that scratch wound induced a sharp 20% rise in astrocyte Ca(2+) that dissipated with distance from the wound. Treatment with IRTX both slowed and dramatically reduced the scratch-induced Ca(2+) increase. Both CPZ and IRTX influenced astrocyte cytoskeletal organization, especially near the wound edge. Taken together, our results indicate that astrocyte mobilization in response to mechanical stress involves influx of extracellular Ca(2+) and cytoskeletal changes in part mediated by TRPV1 activation.

Differential regulation of peripheral IL-1β-induced mechanical allodynia and thermal hyperalgesia in rats

This study examined the differential mechanisms of mechanical allodynia and thermal hyperalgesia after injection of interleukin (IL) 1β into the orofacial area of male Sprague-Dawley rats. The subcutaneous administration of IL-1β produced both mechanical allodynia and thermal hyperalgesia. Although a pretreatment with iodoresiniferatoxin (IRTX), a transient receptor potential vanilloid 1 (TRPV1) antagonist, did not affect IL-1β-induced mechanical allodynia, it significantly abolished IL-1β-induced thermal hyperalgesia. On the other hand, a pretreatment with D-AP5, an N-methyl-d-aspartate (NMDA) receptor antagonist, and NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, blocked IL-1β-induced mechanical allodynia. Pretreatment with H89, a protein kinase A (PKA) inhibitor, blocked IL-1β-induced mechanical allodynia but not thermal hyperalgesia. In contrast, pretreatment with chelerythrine, a protein kinase C (PKC) inhibitor, inhibited IL-1β-induced thermal hyperalgesia. Subcutaneous injections of 2% lidocaine, a local anesthetic agent, blocked IL-1β-induced thermal hyperalgesia but not IL-1β-induced mechanical allodynia. In the resiniferatoxin (RTX)-pretreated rats, a subcutaneous injection of IL-1β did not produce thermal hyperalgesia due to the depletion of TRPV1 in the primary afferent fibers. Double immunofluorescence revealed the colocalization of PKA with neurofilament 200 (NF200) and of PKC with the calcitonin gene-related peptide (CGRP) in the trigeminal ganglion. Furthermore, NMDA receptor 1 (NR1) and TRPV1 predominantly colocalize with PKA and PKC, respectively, in the trigeminal ganglion. These results suggest that IL-1β-induced mechanical allodynia is mediated by sensitized peripheral NMDA/AMPA receptors through PKA-mediated signaling in the large-diameter primary afferent nerve fibers, whereas IL-1β-induced thermal hyperalgesia is mediated by sensitized peripheral TRPV1 receptors through PKC-mediated signaling in the small-diameter primary afferent nerve fibers.

New Transient Receptor Potential Vanilloid Subfamily Member 1 Positron Emission Tomography Radioligands: Synthesis, Radiolabeling, and Preclinical Evaluation

The transient receptor potential vanilloid subfamily member 1 (TRPV1) cation channel is known to be involved in pain nociception and neurogenic inflammation, and accumulating evidence suggests that it plays an important role in several central nervous system (CNS)-related disorders. TRPV1-specific positron emission tomography (PET) radioligands can serve as powerful tools in TRPV1-related (pre)clinical research and drug design. We have synthesized several potent TRPV1 antagonists and accompanying precursors for radiolabeling with carbon-11 or fluorine-18. The cinnamic acid derivative [(11)C]DVV24 and the aminoquinazoline [(18)F]DVV54 were successfully synthesized, and their biological behavior was studied. In addition, the in vivo behavior of a (123)I-labeled analogue of iodo-resiniferatoxin (I-RTX), a well-known TRPV1 antagonist, was evaluated. The binding affinities of DVV24 and DVV54 for human TRPV1 were 163 ± 28 and 171 ± 48 nM, respectively. [(11)C]DVV24, but not [(18)F]DVV54 or (123)I-RTX, showed retention in the trigeminal nerve, known to abundantly express TRPV1. Nevertheless, it appears that ligands with higher binding affinities will be required to allow in vivo imaging of TRPV1 via PET.

Use and limitations of three TRPV-1 receptor antagonists on smooth muscles of animals and man: A vote for BCTC

Specificity of the effect is a crucial factor in using antagonists for detecting the physiological/pathophysiological roles of receptors. Here we examined the capsaicin receptor antagonist effects of three commercially-available substances, capsazepine, iodo-resiniferatoxin (I-RTX) and BCTC, on isolated smooth muscle preparations, including the human intestine. Care was taken to observe possible non-specific effects, to find out safe and effective concentrations. Capsazepine appeared to have a low margin of safety. I-RTX (up to 1μM) specifically inhibited capsaicin-induced contractions in the guinea-pig ileum and urinary bladder. I-RTX showed agonist activity on the rat urinary bladder. BCTC (1μM) abolished the contractile effects of capsaicin (1 or 2μM) on all preparations tested (guinea-pig ileum, bladder, trachea, as well as rat and mouse bladder), and on the guinea-pig renal pelvis, where it failed to influence capsaicin-sensitive, sensory neuron-mediated positive inotropy in response to field stimulation. On human intestinal preparations BCTC prevented the relaxant effect of capsaicin. It is concluded that of the three antagonists tested BCTC seems the safest one for inhibiting TRPV-1 receptors. The effect of capsazepine may be complicated by non-specific inhibition of smooth muscle contractility and that of I-RTX by agonist activity. The “local efferent” function of capsaicin-sensitive sensory neurons is not influenced by BCTC, as shown by the results obtained in the renal pelvis. In conclusion, of the TRPV-1 receptor antagonists studied, BCTC (1μM) seems the most reliable in isolated organ experiments. This substance is also effective in the human intestine.

Inhibitory effects of TRPV1 blocker on UV-induced responses in the hairless mice

The transient receptor potential vanilloid 1 (TRPV1) channel can be activated by vanilloids, exposure to ultraviolet (UV) irradiation, heat, or protons, and conditions that occur during tissue injury. In the present study, we investigated whether or not TRPV1-specific blocker, 5'-iodoresiniferatoxin (I-RTX), can reduce UV-induced matrix metalloproteinases (MMPs), pro-inflammatory cytokines, cyclooxygenase (COX)-2, and p53 expression in the skin of hairless mice. Our results showed that I-RTX inhibited UV-induced skin thickening, as measured by a caliper, or in hematoxylin and eosin (H&E)-stained sections. UV-induced mRNA and protein expression of MMP-13, MMP-9, MMP-3, and MMP-2 was significantly reduced by I-RTX. We also observed the inhibitory effects of I-RTX on UV-induced mRNA expression of the pro-inflammatory cytokines, interleukin (IL)-1β, IL-2, IL-4, and tumor necrosis factor-α. UV-induced COX-2 and p53 protein expression was also significantly decreased by I-RTX. From the above results, we suggest that TRPV1-specific blocker, I-RTX, could prevent UV-induced skin responses, and provide new insight into development of effective therapeutic methods for photoaging.

Participation of the spinal TRPV1 receptors in formalin-evoked pain transduction: a study using a selective TRPV1 antagonist, iodo-resiniferatoxin

The involvement of spinal transient receptor potential vanilloid 1 (TRPV1) in formalin-evoked pain has remained unclear, because investigation of this kind of pain with selective antagonists has not been conducted. The purpose of this study is to investigate the participation of spinal TRPV1 in formalin-evoked pain with iodo-resiniferatoxin (I-RTX), a potent TRPV1-selective antagonist. I-RTX given intrathecally dose-dependently and significantly decreased the number of flinching responses in the formalin-evoked 1st and 2nd phase with ID50 values (drug dose producing 50% inhibition of response) of 1.0 and 3.8 microg, respectively, and concentration-dependently suppressed capsaicin-evoked calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release from rat spinal cord slices with an IC50 value (drug concentration producing 50% inhibition of response) of 86 nM. Capsazepine, a classical non-selective TRPV1 antagonist, given intrathecally also inhibited formalin-evoked flinching in both the 1st and 2nd phase with ID50s of 420 and 200 microg, respectively, and CGRP-LI release from rat spinal cord slices with an IC50 of 7.8 microM. Ratios of in-vivo analgesic potencies of I-RTX and capsazepine well reflected their intrinsic in-vitro activity. These findings suggest that spinal TRPV1 participates in the transduction system of formalin-evoked pain.

The role of transient receptor potential vanilloid-1 on neural responses to acids by the chorda tympani, glossopharyngeal and superior laryngeal nerves in mice

The transient receptor potential vanilloid-1 (TRPV1) receptor acts as a polymodal nociceptor activated by capsaicin, heat, and acid. TRPV1, which is expressed in sensory neurons innervating the oral cavity, is associated with an oral burning sensation in response to spicy food containing capsaicin. However, little is known about the involvement of TRPV1 in responses to acid stimuli in either the gustatory system or the general somatosensory innervation of the oropharynx. To test this possibility, we recorded electrophysiological responses to several acids (acetic acid, citric acid and HCl) and other taste stimuli from the mouse chorda tympani, glossopharyngeal and superior laryngeal nerves, and compared potential effects of iodo-resiniferatoxin (I-RTX), a potent TRPV1 antagonist, on chemical responses of the three nerves. The results indicated that in the chorda tympani nerve, I-RTX (1–100 nM) did not affect responses to acids, sucrose and quinine HCl, but reduced responses to NaCl (I-RTX at concentrations of 10 and 100 nM) and KCl and NH4Cl (100 nM). In contrast, in the glossopharyngeal nerve, I-RTX significantly suppressed responses to all acids and salts, but not to sucrose and quinine HCl. Responses to acetic acid were suppressed by I-RTX even at 0.1 nM concentration. The superior laryngeal nerve responded in a concentration-dependent manner to acetic acid, citric acid, HCl, KCl, NH4Cl and monosodium l-glutamate. The responses to acetic acid, but not to the other stimuli, were significantly inhibited by I-RTX. These results suggested that TRPV1 may be involved in the mechanism for responses to acids presented to the posterior oral cavity and larynx. This high degree of responsiveness to acetic acid may account for the oral burning sensation, known as a flavor characteristic of vinegar.

Fas-associated factor 1 is a negative regulator in capsaicin induced cancer cell apoptosis

Vanilloid receptor1 (VR1/TRPV1) is expressed on peripheral nerves and involved in sensing of temperature and pain. Recent reports have demonstrated that tumor cells express TRPV1 and that capsaicin (CP), a ligand for TRPV1, induces apoptosis in cancer cells. The mechanism underlying CP-induced tumor cell apoptosis remains unclear. Here, we investigated the role of TRPV1 in tumor apoptosis using TRPV1-expressing cancer cell lines. We demonstrate that iodo-resiniferatoxin (I-RTX), an antagonist of TRPV1 does not inhibit CP mediated apoptosis nor is it cytotoxic by itself, but acts as a partial agonist and shows synergistic effect with CP. We further demonstrate that CP treatment degrades Fas-associated factor1 (FAF1); a TRPV1 associated protein. Moreover, using RNA interference with small inhibitory RNAs (siRNA) for FAF1 we observed that down-regulation of FAF1 by siRNA makes the cell susceptible to enhanced apoptosis with CP. In summary, our data shows for the first time that the underlying mechanisms of CP-induced cancer cell apoptosis involves FAF1, a TRPV1 associated protein and serves as an important foundation for further understanding of anticancer activity of CP.

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.

Contribution of TRPV1 to Microglia-Derived IL-6 and NFκB Translocation with Elevated Hydrostatic Pressure

The authors investigated the contributions of the transient receptor potential vanilloid-1 receptor (TRPV1) and Ca(2+) to microglial IL-6 and nuclear factor kappa B (NFkappaB) translocation with elevated hydrostatic pressure. The authors first examined IL-6 colocalization with the microglia marker Iba-1 in the DBA/2 mouse model of glaucoma to establish relevance. They isolated microglia from rat retina and maintained them at ambient or elevated (+70 mm Hg) hydrostatic pressure in vitro and used ELISA and immunocytochemistry to measure changes in the IL-6 concentration and NFkappaB translocation induced by the Ca(2+) chelator EGTA, the broad-spectrum Ca(2+) channel inhibitor ruthenium red, and the TRPV1 antagonist iodo-resiniferatoxin (I-RTX). They applied the Ca(2+) dye Fluo-4 AM to measure changes in intracellular Ca(2+) at elevated pressure induced by I-RTX and confirmed TRPV1 expression in microglia using PCR and immunocytochemistry. In DBA/2 retina, elevated intraocular pressure increased microglial IL-6 in the ganglion cell layer. Elevated hydrostatic pressure (24 hours) increased microglial IL-6 release, cytosolic NFkappaB, and NFkappaB translocation in vitro. These effects were reduced substantially by EGTA and ruthenium red. Antagonism of TRPV1 in microglia partially inhibited pressure-induced increases in IL-6 release and NFkappaB translocation. Brief elevated pressure (1 hour) induced a significant increase in microglial intracellular Ca(2+) that was partially attenuated by TRPV1 antagonism. Elevated pressure induces an influx of extracellular Ca(2+) in retinal microglia that precedes the activation of NFkappaB and the subsequent production and release of IL-6 and is at least partially dependent on the activation of TRPV1 and other ruthenium red-sensitive channels.

Tonic endovanilloid facilitation of glutamate release in brainstem descending antinociceptive pathways.

Activation of transient receptor potential vanilloid-1 (TRPV1) channels in the periaqueductal gray (PAG) activates OFF antinociceptive neurons of the rostral ventromedial medulla (RVM). We examined in rats the effect of intra-ventrolateral (VL)-PAG injections of TRPV1 agonists and antagonists on the nocifensive response to heat in the plantar test, neurotransmitter (glutamate and GABA) release in the RVM, and spontaneous and tail flick-related activities of RVM neurons. The localization of TRPV1 in VL-PAG and RVM neurons was examined using various markers of glutamatergic and GABAergic neurons. Intra-VL-PAG injection of capsaicin increased the threshold of thermal pain sensitivity, whereas the selective TRPV1 antagonist 5'-iodo-resiniferatoxin (I-RTX) facilitated nociceptive responses, and blocked capsaicin analgesic effect at a dose inactive per se. Intra-VL PAG capsaicin evoked a robust release of glutamate in RVM microdialysates. I-RTX, at a dose inactive per se, blocked the effect of capsaicin, and inhibited glutamate release at a higher dose. Antinociception and hyperalgesia induced by capsaicin and I-RTX, respectively, correlated with enhanced or reduced activity of RVM OFF cells. Immunohistochemical analyses suggested that several TRPV1-immunoreactive (ir) neurons in both the VL-PAG and RVM are glutamatergic and surrounded by glutamatergic and GABAergic terminals. Our data suggest that VL-PAG neurons respond to TRPV1 stimulation by releasing glutamate into the RVM, thereby activating OFF cells and producing analgesia. The results obtained with the TRPV1 antagonist alone suggest that this pathway is tonically activated by endovanilloids.

Animal models of cough: Literature review and presentation of a novel cigarette smoke-enhanced cough model in the guinea-pig

A wealth of literature describes the approaches that investigators have used to develop animal models of cough. The relevance of the models to cough in man and disease is still unknown. Furthermore, the choice of animal model that is used will depend on the purpose of the investigation and what questions are being asked. Cigarette smoke is known to cause COPD and cough is a principle symptom where patients demonstrate an increased cough response to citric acid or capsaicin. This paper describes the development of exacerbated cough to these agents in the guinea-pig following cigarette smoke exposure and pharmacological profiling of these models. Male Dunkin–Hartley guinea-pigs were exposed to air or cigarette smoke (4 or 5 research cigarettes daily for the capsaicin and citric acid studies, respectively) for a 3 s puff every 30 s, for up to 10 days. At selected time points conscious, unrestrained animals were placed in a plethysmograph chamber and challenged with an aerosol of 0.3 M citric acid (10 min) or 10 μM capsaicin (7 min). Cough and Penh area under the curve (AUC) were recorded during the exposure and for a further 10 min (citric acid) or 8 min (capsaicin) after exposure. Compounds were administered on day 3 or 11 for citric acid or capsaicin, respectively. Significant enhancement of citric acid-induced cough was evident 24 h (12±2 to 24±4 * coughs) after a single exposure and further enhanced after 2 days (13±3 to 36±4 * coughs). Enhanced cough to capsaicin was not reliable until after 10 days of cigarette smoke exposure (2±1 to 14±3** coughs). Data are expressed as mean±s.e.mean ( n=10), * p<0.05, ** p<0.01 vs. air-exposed animals (Mann–Whitney rank-sum test). The minimum effective doses to inhibit citric acid-induced cough were 10, 10, 3 and 0.3 mg/kg for codeine (p.o. −30 min), a selective NK 1/NK 2 antagonist, DNK333 (p.o. −2 h), terbutaline (s.c. −1 h) and atropine (s.c. −1 h), respectively. The minimum effective doses to inhibit capsaicin-induced cough were 3, 1, 0.3 and 0.3 mg/kg for codeine, DNK333, terbutaline (p.o. −2 h) and atropine, respectively. The VR1 antagonists capsazepine and iodo-resiniferatoxin (IRTX) did not inhibit cough in either model. Differences in sensitivity between citric acid and capsaicin to pharmacological agents may be partly explained by the difference in magnitude of response to these agents. Clinically used compounds such as codeine and terbutaline have shown activity in both models, however the relevance of the models to cough in man and disease for potential new therapies is unknown.

Transient Receptor Potential Vanilloid Subtype 1 Mediates Microglial Cell Death In Vivo and In Vitro via Ca2+-Mediated Mitochondrial Damage and CytochromecRelease

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.

Functional Mapping of the Transient Receptor Potential Vanilloid 1 Intracellular Binding Site

Capsaicin (vanilloid) sensitivity has long served as the functional signature of a subset of nociceptive sensory neurons. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 547 (transmembrane region 4) mediates significant species differences in resiniferatoxin (RTX) sensitivity, and the Ser(512) residue is critical in discriminating between pH and capsaicin gating. In the present study, the pharmacological profiles of a variety of ligands were studied to investigate cross-talk between these two regions. Exchange of residue 547 between species mediated a difference in capsaicin and RTX-dependent gating. Likewise, the potency of iodoresiniferatoxin (I-RTX) and a novel transient receptor potential vanilloid 1 antagonist were also altered. Experiments using the S512Y mutant channel have confirmed the importance of residue 512 for functional interaction of capsaicin and our novel antagonist. In this study, we were surprised to find that the mutation S512Y converted the activity of the antagonist I-RTX into an intrinsic agonist, albeit with a lower potency than its parent compound, RTX. Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the voltage-dependent potassium channel, in which both the 512 and 547 amino acid residues are in close proximity. Our data support the model whereby intracellular ligand interaction occurs within an S3-S4 "sensor" domain, enabling binding of ligands to be transduced to functional gating of the channel. The binding pocket also seems to be exquisitely sensitive to residue-specific interaction with ligands, because subtle changes in either ligand or channel structure can have profound effects on channel activity.

Enhancement of Spontaneous and Heat-Evoked Activity in Spinal Nociceptive Neurons by the Endovanilloid/Endocannabinoid N-Arachidonoyldopamine (NADA)

N-arachidonoyldopamine (NADA) is an endogenous molecule found in the nervous system that is capable of acting as a vanilloid agonist via the TRPV1 receptor and as a cannabinoid agonist via the CB1 receptor. Using anesthetized rats, we investigated the neural correlates of behavioral thermal hyperalgesia produced by NADA. Extracellular single cell electrophysiology was conducted to assess the effects of peripheral administration of NADA (i.pl.) on nociceptive neurons in the dorsal horn of the spinal cord. Injection of NADA in the hindpaw caused increased spontaneous discharge of spinal nociceptive neurons compared with injection of vehicle. The neurons also displayed magnified responses to application of thermal stimuli ranging from 34 to 52 degrees C. NADA-induced neural hypersensitivity was dose dependent (EC50 = 1.55 microg) and TRPV1 dependent, as the effect was abolished by co-administration of the TRPV1 antagonist 5'-iodoresiniferatoxin (I-RTX). In contrast, co-administration of the CB1 antagonist SR 141716A did not attenuate this effect. These results suggest that the enhanced responses of spinal nociceptive neurons likely underlie the behavioral thermal hyperalgesia and implicate a possible pain-sensitizing role of endogenous NADA mediated by TRPV1 in the periphery.

Inhibition of peripheral vanilloid TRPV1 receptors reduces noxious heat‐evoked responses of dorsal horn neurons in naïve, carrageenan‐inflamed and neuropathic rats

The vanilloid TRPV1 receptor, present on primary afferent fibres, is activated by noxious heat, low pH and endogenous vanilloids. Changes in the function or distribution of TRPV1 receptors may play an important role in pain induced by inflammation or neuropathy. The aim of the present study was to evaluate the role of peripheral TRPV1 receptors in thermal nociception in rat models of inflammatory and neuropathic pain. Here, we have determined the effects of peripheral administration of the potent TRPV1 receptor antagonist iodoresiniferatoxin (IRTX) on noxious heat (45 degrees C)-evoked responses of spinal wide dynamic range (WDR) neurons in naïve, carrageenan-inflamed, sham-operated and L5/6 spinal nerve-ligated (SNL) anaesthetized rats in vivo. In addition, effects of peripheral administration of IRTX on mechanically evoked responses of WDR neurons were determined in sham-operated and SNL rats. Carrageenan inflammation significantly (P<0.05) increased the 45 degrees C-evoked responses of WDR neurons. Intraplantar injection of the lower dose of IRTX (0.004 microg) inhibited (P<0.05) 45 degrees C-evoked responses of WDR neurons in carrageenan-inflamed, but not in naïve, rats. The higher dose of IRTX (0.4 microg) significantly (P<0.05) inhibited 45 degrees C-evoked responses in both inflamed and naïve rats. In sham-operated and SNL rats, IRTX (0.004 and 0.4 microg) significantly (P<0.05) inhibited 45 degrees C-evoked, but had no effect on mechanically evoked responses of WDR neurons. These data support the role of peripheral TRPV1 receptors in noxious thermal transmission in naïve, inflamed and neuropathic rats, and suggest that there is an increased functional contribution of peripheral TRPV1 receptors following acute inflammation.