Resiniferatoxin Research Articles

Lipid-Mediated Interaction of Double-Knot Toxin with TRPV1 Channels

The Transient Receptor Potential V1 (TRPV1) channel is a cation channel that opens in response to diverse chemical and physical stimuli. These stimuli include capsaicin, temperature, pH and bioactive lipids. Recently, a novel peptide named Double cystine Knot Toxin (DkTx) was purified from tarantula venom and shown to activate the channel from the extracellar site of the membrane and proposed to bind the pore region (Bohlen CJ., et al, 2010). In order to characterize DkTx-TRPV1 interactions, we expressed and purified recombinant His-tagged DkTx in E. coli and 1D4-tagged TRPV1 in Saccharomyces cerevisiae. Expressed TRPV1 binds the specific TRPV1 agonist, 3H- resiniferatoxin (RTx), with high affinity (Kd∼500 pM) and in the presence of DkTx the affinity of RTx increases by ∼4-fold, indicating that both RTx and DkTx interact with the TRPV1 channel in membranes, but at distinct sites that interact allosterically. Pull-down experiments with full-length and truncated TRPV1 (containing the pore-forming S5-S6 segments and the C-terminus suggest that DkTx interacts with both detergent purified channel constructs, supporting an interaction of the toxin with the external pore region. DkTx and TRPV1 constructs do not, however, co-elute as a complex in size-exclusion or affinity chromatography, suggesting that in detergent solution the interactions are weak. To investigate the strength of TRPV1 interactions with DkTx in detergent micelles we utilized surface plasmon resonance with TRPV1 deposited on a gold chip using 1D4 tag. DkTx interacts with TRPV1 in detergent micelles, but with much lower apparent affinity than observed in membranes. The isolated K2 lobe of DkTx exhibited higher affinity than K1, in agreement with the previous electrophysiology studies. These results support the notion that DkTx interacts with the external pore of TRPV1 and suggest that the membrane lipids play a critical role in toxin-channel interaction.

Influence of urethane-anesthesia on the effect of resiniferatoxin treatment on bladder function in rats with spinal cord injury

We investigated the effect of resiniferatoxin (RTX)-treatment on cystometric parameters in the spinal cord injury (SCI) rats in both conscious and urethane-anesthetized conditions and evaluated the influence of urethane-anesthesia on the effect of RTX on lower urinary tract (LUT) function in SCI rats. Female Sprague-Dawley rats were used. SCI was created by transection of the T8-T9 spinal cord. Four weeks after the transection, the animals were placed in a restraint cage for the first cystometric measurements in a conscious state. Secondary cystometric measurements were performed in a conscious condition following the 1 day after RTX-(0.3 mg/kg) or vehicle-subcutaneous injection. Then the animals were injected with urethane (1.5 g/kg, subcutaneously), and cystometric measurements were repeated four times every 1 hr-interval. After the RTX-treatment in a conscious condition, urinary retention was observed in three out of five animals. In addition, the number of non-voiding contractions (NVCs) significantly decreased although their amplitude did not change significantly. After the urethane-injection, all of the animals treated with RTX developed urinary retention. The amplitude of NVCs significantly decreased, whereas the number of NVCs did not change significantly in the RTX-treated group. No cystometric parameters significantly changed after either vehicle- or urethane-injection in the vehicle-treated group. The present results indicate that the suppressive effects of RTX on NVCs as well as voiding contractions in SCI rats can be enhanced by urethane-anesthesia. Such suppressive effect of urethane-anesthesia itself should be taken into consideration when we evaluate a drug-effect on LUT function in rats with SCI.

Intravesical Resiniferatoxin for the Treatment of Storage Lower Urinary Tract Symptoms in Patients with Either Interstitial Cystitis or Detrusor Overactivity: A Meta-Analysis

BackgroundWhile Resin­iferatoxin (RTX) has been widely used for patients with storage lower urinary tract symptoms (LUTS), its clinical efficiency hasn't yet been well evaluated. A meta-analysis was performed to evaluate the exact roles of intravesical RTX for the treatment of storage LUTS in patients with either interstitial cystitis (IC) or detrusor overactivity (DO).MethodsA meta-analysis of RTX treatment was performed through a comprehensive search of the literature. In total, 2,332 records were initially recruited, 1,907 from Elsevier, 207 from Medline and 218 from the Web of Science. No records were retrieved from the Embase or Cochrane Library. Seven trials with 355 patients were included and one trial was excluded because of the lack of extractable data. The analyses were all performed using RevMan 5.1 and MIX 2.0.ResultsBladder pain was significantly reduced after RTX therapy in patients with either IC or DO. The average decrease of the visual an alogue pain scale was 0.42 after RTX treatment (p = 0.02). The maximum cystometric capacity (MCC) was significantly increased in patients with DO (MCC increase, 53.36 ml, p = 0.006) but not in those with IC (MCC increase, −19.1 ml, p = 0.35). No significant improvement in urinary frequency, nocturia, incontinence or the first involuntary detrusor contraction (FDC) was noted after RTX therapy (p = 0.06, p = 0.52, p = 0.19 and p = 0.41, respectively).ConclusionsRTX could significantly reduce bladder pain in patients with either IC or DO, and increase MCC in patients with DO; however, no significant improvement was observed in frequency, nocturia, incontinence or FDC. Given the limitations in the small patient size and risk of bias in the included trials, great caution should be taken when intravesical RTX is used before a large, multicenter, well-designed random control trial with a long-term follow-up is carried out to further assess the clinical efficacy of RTX in in patients with storage LUTS.

Depressive behavior in the forced swim test can be induced by TRPV1 receptor activity and is dependent on NMDA receptors

Blocking, desensitizing, or knocking out transient receptor potential vanilloid type 1 (TRPV1) receptors decreases immobility in the forced swim test, a measure of depressive behavior. We questioned whether enhancing TRPV1 activity promotes immobility in a fashion that is prevented by antidepressants. To test this we activated heat-sensitive TRPV1 receptors in mice by water that is warmer than body temperature (41°C) or a low dose of resiniferatoxin (RTX). Water at 41°C elicited less immobility than cooler water (26°C), indicating that thermoregulatory sites do not contribute to immobility. Although a desensitizing regimen of RTX (3–5 injections of 0.1mg/kg s.c.) decreased immobility during swims at 26°C, it did not during swims at 41°C. In contrast, low dose of RTX (0.02mg/kg s.c.) enhanced immobility, but only during swims at 41°C. Thus, activation of TRPV1 receptors, endogenously or exogenously, enhances immobility and these sites are activated by cold rather than warmth. Two distinct types of antidepressants, amitriptyline (10mg/kg i.p.) and ketamine (50mg/kg i.p.), each inhibited the increase in immobility induced by the low dose of RTX, verifying its mediation by TRPV1 sites. When desensitization was limited to central populations using intrathecal injections of RTX (0.25μg/kg i.t.), immobility was attenuated at both temperatures and the increase in immobility produced by the low dose of RTX was inhibited. This demonstrates a role for central TRPV1 receptors in depressive behavior, activated by conditions (cold stress) distinct from those that activate TRPV1 receptors along thermosensory afferents (heat).

Resiniferatoxin (RTX) Causes a Uniquely Protracted Musculoskeletal Hyperalgesia in Mice by Activation of TRPV1 Receptors

Inactivation of transient receptor potential vanilloid-1 (TRPV1) receptors is one approach to analgesic drug development. However, TRPV1 receptors exert different effects on each modality of pain. Because muscle pain is clinically important, we compared the effect of TRPV1 ligands on musculoskeletal nociception to that on thermal and tactile nociception. Injected parenterally, capsaicin had no effect on von Frey fiber responses (tactile) but induced a transient hypothermia and hyperalgesia in both the tail flick (thermal) and grip force (musculoskeletal) assays, presumably by its agonistic action at TRPV1 sites. In contrast, resiniferatoxin (RTX) produced a chronic (>58 days) thermal antinociception, consistent with its reported ability to desensitize TRPV1 sites. In the same mice, RTX produced a transient hypothermia (7 hours) and a protracted (28-day) musculoskeletal hyperalgesia in spite of a 35.5% reduction in TRPV1 receptor immunoreactivity in muscle afferents. Once musculoskeletal hyperalgesia subsided, mice were tolerant to the hyperalgesic effects of either capsaicin or RTX whereas tolerance to hypothermia did not develop until after 3 injections. Musculoskeletal hyperalgesia was prevented but not reversed by SB-366791, a TRPV1 antagonist, indicating that TRPV1 receptors initiate but do not maintain hyperalgesia. Injected intrathecally, RTX produced only a brief musculoskeletal hyperalgesia (2 days), after which mice were tolerant to this effect. PerspectiveThe effect of TRPV1 receptors varies depending on modality and tissue type, such that RTX causes thermal antinociception, musculoskeletal hyperalgesia, and no effect on tactile nociception in healthy mice. Spinal TRPV1 receptors are a potential target for pain relief as they induce only a short musculoskeletal hyperalgesia followed by desensitization.

Additive antiemetic efficacy of low-doses of the cannabinoid CB1/2 receptor agonist Δ9-THC with ultralow-doses of the vanilloid TRPV1 receptor agonist resiniferatoxin in the least shrew (Cryptotis parva)

Previous studies have shown that cannabinoid CB1/2 and vanilloid TRPV1 agonists (delta-9-tetrahydrocannabinol (Δ9-THC) and resiniferatoxin (RTX), respectively) can attenuate the emetic effects of chemotherapeutic agents such as cisplatin. In this study we used the least shrew to demonstrate whether combinations of varying doses of Δ9-THC with resiniferatoxin can produce additive antiemetic efficacy against cisplatin-induced vomiting. RTX by itself caused vomiting in a bell-shaped dose-dependent manner with maximal vomiting at 18μg/kg when administered subcutaneously (s.c.) but not intraperitoneally (i.p.). Δ9-THC up to 10mg/kg provides only 80% protection of least shrews from cisplatin-induced emesis with an ID50 of 0.3–1.8mg/kg. Combinations of 1 or 5μg/kg RTX with varying doses of Δ9-THC completely suppressed both the frequency and the percentage of shrews vomiting with ID50 dose values 5–50 times lower than Δ9-THC doses tested alone against cisplatin. A less potent TRPV1 agonist, capsaicin, by itself did not cause emesis (i.p. or s.c.), but it did significantly reduce vomiting induced by cisplatin given after 30min but not at 2h. The TRPV1-receptor antagonist, ruthenium red, attenuated cisplatin-induced emesis at 5mg/kg; however, another TRPV1-receptor antagonist, capsazepine, did not. In summary, we present evidence that combination of CB1/2 and TRPV1 agonists have the capacity to completely abolish cisplatin-induced emesis at doses that are ineffective when used individually.

Abstract 536: Resiniferatoxin-induced Disorders in Glucose Homeostasis and Hypertension: Role of Trpv1-positive Sensory Nerves

Resiniferatoxin (RTX), an ultrapotent agonist of the transient receptor potential vanilloid 1 (TRPV1) channels, has been used to relieve pain in humans in clinical trials that are ongoing, completed, or terminated due to its unwanted side effects that have not been well understood. We test the hypothesis that RTX causes disorders in glucose homeostasis, renal functional and structural impairment, and hypertension via insulin receptor-mediated increases in sympathetic nerve activity. Male Wistar rats were injected with RTX (0.2 mg/kg, i.p.) or vehicle. Pancreatic and renal TRPV1 levels were decreased 4 weeks after RTX treatment, confirming effective and lasting treatment of RTX (p<0.05). RTX decreased plasma insulin levels (Control: 1.4±0.1 vs RTX: 0.8±0.1 ng/ml, p<0.05) but improved glucose tolerance (p<0.05). RTX increased the translocation of skeletal muscle glucose transporter 4 (Glut4) from cytosol to cell membrane indicated by increased membrane fraction but decreased cytosol fraction of Glut4 in RTX-treated rats (p<0.05). RTX increased levels of phosphorylated insulin receptor substrate 1 in sympathetic ganglia (Control: 0.43±0.04 vs RTX: 0.65±0.08, p<0.05), renal tyrosine hydroxylase contents, and urinary norepinephrine concentrations (Control: 352±70 vs RTX: 781±142 ng/day, p<0.05). When TRPV1 expressed in sensory neurons and their nerve endings innervating the kidney was knocked down by intrathecal (i.t) injection of TRPV1 shRNA, insulin (3nmol/kg, i.t.)-induced increases in efferent renal nerve activity were further enhanced (Control: 40±3 vs TRPV1 shRNA: 53±2, p<0.05). RTX decreased creatinine clearance (Control: 0.58±0.04 vs RTX: 0.46±0.04 ml/min/100 gbwt, p<0.05) and increased levels of plasma urea, urinary albumin, renal connective tissue growth factor, and systolic blood pressure (Control: 128±1 vs RTX: 143±2 mmHg, p<0.05). Thus, RTX treatment causes disorders in glucose homeostasis, renal functional and structural impairment, and hypertension, possibly via increasing sympathetic nerve activity resulting from enhanced stimulatory effects of the insulin receptors expressed in sympathetic nerves and weakened inhibitory effects of TRPV1-positive sensory nerves on sympathetic nerves.

The Role of Nerve Growth Factor in Neuropathic Pain Inhibition Produced by Resiniferatoxin Treatment in the Dorsal Root Ganglia

Resiniferatoxin (RTX), an excitotoxic agonist for vanilloid receptor 1, is a promising candidate for intractable pain treatment. We evaluated the effects of RTX, applied to dorsal root ganglia (DRG) at high doses (1200 ng), in sensory-motor function and nerve growth factor (NGF) alterations in a photochemical sciatic nerve injury rat model. Following RTX injection into the L3-6 DRG at high doses and behavioral evaluation, the rats were sacrificed and the DRG were tested by immunohistochemistry and mRNA analysis for NGF and its' receptors, tyrosine kinase A (TrkA) and p75. The correlation between neuropathic pain and NGF, TrkA, and p75 expression was analyzed. The treated rats had preserved touch, cold, pain, and high-heat sensations, and exhibited hypoalgesia to low-heat stimulation. After RTX treatment, TrkA and p75 altered their expressions from one neuronal type to another in the DRG. NGF and p75 expression changed from the small-size neurons in neuropathic rat DRG to the large- and medium-size neurons in non-neuropathic and RTX-treated animals, concomitantly with neuropathic pain suppression. TrkA was expressed in the small-size neurons in neuropathic rat DRG, and was drastically reduced in all size neurons after RTX treatment. NGF, TrkA, and p75 mRNA expression supported these phenotypic changes in the DRG. The pathway of NGF-TrkA expressed in the small-size neurons, associated with neuropathic pain, was shifted to the NGF-p75 pathway expressed in the large-size neurons after RTX treatment, in association with neuropathic pain inhibition. These findings may play an important role in clinical trial designs.

Identification of the sensory nerve fiber responsible for lysophosphatidic acid-induced allodynia in mice

Lysophosphatidic acid (LPA) has been considered one of the molecular culprits for neuropathic pain. Understanding how LPA changes the function of primary afferent fibers might be an essential step for clarifying the pathogenesis of neuropathic pain. The present study was designed to identify the primary afferent fibers (Aβ, Aδ, or C) participating in LPA-induced allodynia in ddY mice. Mechanical allodynia and thermal hyperalgesia were evaluated by the von Frey filament test and thermal paw withdrawal test, respectively. Sensory nerve fiber responsiveness was measured using a Neurometer. Daily repeated intrathecal treatment with LPA led to a decrease in the mechanical, but not thermal nociceptive threshold, and a reduction in the threshold for paw withdrawal induced by 2000-Hz (Aβ fiber) and 250-Hz (Aδ fiber), but not 5-Hz (C fiber) sine-wave electrical stimulation. When the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor agonist resiniferatoxin (RTX) was administered subcutaneously before the start of LPA treatment, LPA-induced mechanical allodynia and Aβ and Aδ fiber hypersensitivity demonstrated by neurometry were not affected, indicating that TRPV1-expressing nerve fibers (possibly C fibers) might not be essential for LPA-induced allodynia. LPA-induced allodynia was reversed by treatment with RTX at 7days after the start of LPA treatment. Expression of TRPV1 on myelinated nerve fibers after repeated intrathecal LPA treatment was observed in the dorsal root ganglion. These results suggest that sensitization of Aβ and Aδ fibers, but not C fibers, contributes to the development of intrathecally administered LPA-induced mechanical allodynia. Moreover, increased or newly expressed TRPV1 receptors in Aβ and Aδ fibers are considered to be involved in the maintenance of LPA-induced allodynia.

TRPM5-dependent amiloride- and benzamil-insensitive NaCl chorda tympani taste nerve response

Transient receptor potential (TRP) subfamily M member 5 (TRPM5) cation channel is involved in sensing sweet, bitter, umami, and fat taste stimuli, complex-tasting divalent salts, and temperature-induced changes in sweet taste. To investigate if the amiloride- and benzamil (Bz)-insensitive NaCl chorda tympani (CT) taste nerve response is also regulated in part by TRPM5, CT responses to 100 mM NaCl + 5 μM Bz (NaCl + Bz) were monitored in Sprague-Dawley rats, wild-type (WT) mice, and TRP vanilloid subfamily member 1 (TRPV1) and TRPM5 knockout (KO) mice in the presence of resiniferatoxin (RTX), a TRPV1 agonist. In rats, NaCl + Bz + RTX CT responses were also monitored in the presence of triphenylphosphine oxide, a specific TRPM5 blocker, and capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791), specific TRPV1 blockers. In rats and WT mice, RTX produced biphasic effects on the NaCl + Bz CT response, enhancing the response at 0.5-1 μM and inhibiting it at >1 μM. The NaCl + Bz + SB-366791 CT response in rats and WT mice and the NaCl + Bz CT response in TRPV1 KO mice were inhibited to baseline level and were RTX-insensitive. In rats, blocking TRPV1 by capsazepine or TRPM5 by triphenylphosphine oxide inhibited the tonic NaCl + Bz CT response and shifted the relationship between RTX concentration and the magnitude of the tonic CT response to higher RTX concentrations. TRPM5 KO mice elicited no constitutive NaCl + Bz tonic CT response. The relationship between RTX concentration and the magnitude of the tonic NaCl + Bz CT response was significantly attenuated and shifted to higher RTX concentrations. The results suggest that pharmacological or genetic alteration of TRPM5 activity modulates the Bz-insensitive NaCl CT response and its modulation by TRPV1 agonists.

Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors

The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26 °C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor.

Additive antiemetic efficacy of Δ9‐THC with vanilloid TRPV1 receptor agonists in the least shrew (Cryptotis parva)

Previous studies have shown that cannabinoid CB1 and vanilloid TRPV1 agonists ( Δ9‐THC and resiniferatoxin (RTX), respectively) can attenuate the emetic effects of chemotherapeutic agents such as cisplatin. In this study we used the least shrew to demonstrate whether combination of varying doses of Δ9‐THC with resiniferatoxin can produce additive antiemetic efficacy against cisplatin‐induced vomiting. RTX by itself caused vomiting in a bell‐shaped dose‐dependent manner with maximal vomiting at 18 ug/kg when administered sc but not ip. Δ9‐THC up to 10 mg/kg can only protect least shrews from cisplatin‐induced emesis by 80% with an ID50 of 0.3 – 1.8 mg/kg. Combinations of 1 or 5 ug/kg RTX with varying doses of Δ9‐THC completely suppressed both the frequency and the percentage of shrews vomiting with ID50 dose values 5–50 times lower than Δ9‐THC doses tested alone against cisplatin. A less potent TRPV1 agonist capsaicin, by itself did not cause emesis (ip or sc), but did significantly reduce vomiting induced by cisplatin given after 30 minutes but not at 2 hours. TRPV1 antagonist ruthenium red attenuated cisplatin‐induced emesis at 5 mg/kg, however capsazepine did not. In summary, we present evidence that combinations of CB1 and TRPV1 agonists have the capacity to completely abolish cisplatin‐induced emesis at doses lower than when used individually.

Expression and functionality of TRPV1 receptor in human MCF‐7 and canine CF.41 cells

As canine mammary tumours (CMT) and human breast cancer share clinical and prognostic features, the former have been proposed as a model to study carcinogenesis and improved therapeutic treatment in human breast cancer. In recent years, it has been shown that transient receptor potential vanilloid 1 (TRPV1) is expressed in different neoplastic tissues and its activation has been associated with regulation of cancer growth and progression. The aim of the present research was to demonstrate the presence of TRPV1 in human and canine mammary cancer cells, MCF-7 and CF.41, respectively, and to study the role of TRPV1 in regulating cell proliferation. The images obtained by Western blot showed a signal at 100 kDa corresponding to the molecular weight of TRPV1 receptor. All tested TRPV1 agonists and antagonists caused a significant decrease (P < 0.05) of cell growth rate in MCF-7 cells. By contrast, in CF.41 cells capsaicin and capsazepine induced a significant increase (P < 0.05) in cell proliferation, whereas resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX) had no influence on CF.41 cell proliferation. Further studies are needed to elucidate the underlying molecular mechanism responsible for the different effects evoked by TRPV1 activation in MCF-7 and CF.41 cells.

Resiniferatoxin for Pain Treatment: An Interventional Approach to Personalized Pain Medicine.

This review examines existing preclinical and clinical studies related to resiniferatoxin (RTX) and its potential uses in pain treatment. Like capsaicin, RTX is a vanilloid receptor (TRPV1) agonist, only more potent. This increased potency confers both quantitative and qualitative advantages in terms of drug action on the TRPV1 containing nerve terminal, which result in an increased efficacy and a long duration of action. RTX can be delivered by a central route of administration through injection into the subarachnoid space around the lumbosacral spinal cord. It can also be administered peripherally into a region of skin or deep tissue where primary afferents nerves terminate, or directly into a nerve trunk or a dorsal root ganglion. The central route is currently being evaluated as a treatment for intractable pain in patients with advanced cancer. Peripheral administration offers the possibility to treat a wide diversity of pain problems because of the ability to bring the treatment to the site of the pain (the peripheral generator). While not all pain disorders are appropriate for RTX, tailoring treatment to an individual patient's needs via a selective and local intervention that chemically targets a specific population of nerve terminals provides a new capability for pain therapy and a simplified and effective approach to personalized pain medicine.

Astrocytic TRPV1 ion channels detect blood‐borne signals in the sensory circumventricular organs of adult mouse brains

The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) sense a variety of blood-borne molecules because they lack typical blood-brain barrier. Though a few signaling pathways are known, it is not known how endogenous ligands for transient receptor potential vanilloid receptor 1 ion channel (TRPV1) are sensed in the CVOs. In this study, we aimed to examine whether or not astrocytic TRPV1 senses directly blood-borne molecules in the OVLT, SFO, and AP of adult mice. The reverse transcription-polymerase chain reaction and Western analysis revealed the expression of TRPV1 in the CVOs. Confocal microscopic immunohistochemistry further showed that TRPV1 was localized prominently at thick cellular processes of astrocytes rather than fine cellular processes and cell bodies. TRPV1-expressing cellular processes of astrocytes surrounded the vasculature to constitute dense networks. The expression of TRPV1 was also found at neuronal dendrites but not somata in the CVOs. The intravenous administration of a TRPV1 agonist resiniferatoxin (RTX) prominently induced Fos expression at astrocytes in the OVLT, SFO, and AP and neurons in adjacent related nuclei of the median preoptic nuclei (MnPO) and nucleus of the solitary tract (Sol) of wild-type but not TRPV1-knockout mice. The intracerebroventricular infusion of RTX induced Fos expression at both astrocytes and neurons in the CVOs, MnPO, and Sol. Thus, this study demonstrates that blood-borne molecules are sensed directly by astrocytic TRPV1 of the CVOs in adult mammalians.

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.

Antidiuretic effect of antimuscarinic agents in rat model depends on C‐fibre afferent nerves in the bladder

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Antichollnergic agents are anticipated to diminish storage symptoms, as well as nocturia. Nevertheless, the effect of this treatment on polyuria related to nocturia is not clear. By subgroup analysis of the data set from a phase III clinical trial of antimuscarinic agent for OAB patients in Japan, imidafenacin was found to improve nocturia with a reduction in nocturnal polyuria. This study adds the effects and underlying mechanism of antimuscarinic agents decreasing urine production through inhibition of C-fibre in the bladder of water-leaded rats. To evaluate the effects and underlying mechanisms of antimuscarinic agents used to decrease in urine production in water-loaded rats. Urine production was measured using a cystostomy catheter in female Sprague-Dawley rats every 2 h. The effect of the antimuscarinic agents atropine, tolterodine and imidafenacin on urine production was investigated under water-loaded conditions, which were induced by i.p. injection of 15 mL saline. Blood samples were collected to determine the levels of antidiuretic hormone (ADH), aldosterone (ALD), atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) before, and 2 and 8 h after, antimuscarinic agent administration. To induce desensitization of C-fibre afferent nerves, resiniferatoxin (RTX)was injected s.c. or intravesically 2 days before experiments. Urine production increased and reached its maximum 2 h after 15 mL saline injection. Imidafenacin and tolterodine decreased urine production in water-loaded rats, but ADH, ALD, ANP and BNP levels were not different between imidafenacin-treated and vehicle-treated rats. The inhibitory effect on urine production was not found in RTX-treated rats. Atropine did not reduce urine production. These results suggest that antimuscarinic agents decrease urine volume through C-fibres in the bladder; thus, antimuscarinics with inhibitory effects on C-fibres could be beneficial for nocturia with nocturnal polyuria.

Promotion of thermal analgesia and neuropeptidergic skin reinnervation by 4-methylcatechol in resiniferatoxin-induced neuropathy

To investigate whether 4-methylcatechol (4MC) could decrease the duration of the thermosensation disorder and promote the innervation of peptidergic intraepidermal nerve fibers (IENFs), we developed a resiniferatoxin (RTX)-induced neuropathic mouse model with thermal analgesia and skin denervation that was followed by daily 4MC treatment. On day 7 after RTX administration (RTXd7), the substance P (SP)(+) IENFs were completely depleted compared with the vehicle group (p < 0.0001), whereas the calcitonin gene-related peptide (CGRP)(+) IENFs were dramatically, but not completely, depleted (p < 0.0001). While SP(+) IENFs remained depleted (p = 0.0043), CGRP(+) IENFs were recovered by RTXd84 (p = 0.78). 4MC had no effect on the reinnervation of SP(+) IENFs, but markedly promoted the reinnervation of CGRP(+) IENFs on RTXd35 (p = 0.035). On RTXd56, CGRP(+) IENFs were comparable with the vehicle group (p = 0.39). In addition, 4MC normalized thermal analgesia on RTXd35 compared with RTX group (p = 0.007). In the current study, the significant promotion of reinnervation of CGRP(+) IENFs and thermal latencies on RTXd35 by 4MC indicated that CGRP(+) IENFs were responsible for the thermal transmission in chronic phase of RTX-induced neuropathy.

Effects of Herpes Simplex Virus Vector–Mediated Enkephalin Gene Therapy on Bladder Overactivity and Nociception

We previously reported the effects of herpes simplex virus (HSV) vector-mediated enkephalin on bladder overactivity and pain. In this study, we evaluated the effects of vHPPE (E1G6-ENK), a newly engineered replication-deficient HSV vector encoding human preproenkephalin (hPPE). vHPPE or control vector was injected into the bladder wall of female rats 2 weeks prior to the following studies. A reverse-transcription PCR study showed high hPPE transgene levels in L6 dorsal root ganglia innervating the bladder in the vHPPE group. The number of freezing behaviors, which is a nociceptive reaction associated with bladder pain, was also significantly lower in the vHPPE group compared with the control group. The number of L6 spinal cord c-fos-positive cells and the urinary interleukin (IL)-1β and IL-6 levels after resiniferatoxin (RTx) administration into the bladder of the vHPPE group were significantly lower compared with those of the control vector-injected group. In continuous cystometry, the vHPPE group showed a smaller reduction in intercontraction interval after RTx administration into the bladder. This antinociceptive effect was antagonized by naloxone hydrochloride. Thus, the HSV vector vHPPE encoding hPPE demonstrated physiological improvement in visceral pain induced by bladder irritation. Gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Anatomical and functional characterization of a duodeno-pancreatic neural reflex that can induce acute pancreatitis

Neural cross talk between visceral organs may play a role in mediating inflammation and pain remote from the site of the insult. We hypothesized such a cross talk exists between the duodenum and pancreas, and further it induces pancreatitis in response to intraduodenal toxins. A dichotomous spinal innervation serving both the duodenum and pancreas was examined, and splanchnic nerve responses to mechanical stimulation of these organs were detected. This pathway was then excited on the duodenal side by exposure to ethanol followed by luminal mustard oil to activate transient receptor potential subfamily A, member 1 (TRPA1). Ninety minutes later, pancreatic inflammation was examined. Ablation of duodenal afferents by resiniferatoxin (RTX) or blocking TRPA1 by Chembridge (CHEM)-5861528 was used to further investigate the duodeno-pancreatic neural reflex via TRPA1. ~40% of dorsal root ganglia (DRG) from the spinal cord originated from both duodenum and pancreas via dichotomous peripheral branches; ~50% splanchnic nerve single units responded to mechanical stimulation of both organs. Ethanol sensitized TRPA1 currents in cultured DRG neurons. Pancreatic edema and myeloperoxidase activity significantly increased after intraduodenal ethanol followed by mustard oil (but not capsaicin) but significantly decreased after ablation of duodenal afferents by using RTX or blocking TRPA1 by CHEM-5861528. We found the existence of a neural cross talk between the duodenum and pancreas that can promote acute pancreatitis in response to intraduodenal chemicals. It also proves a previously unexamined mechanism by which alcohol can induce pancreatitis, which is novel both in terms of the site (duodenum), process (neurogenic), and receptor (TRPA1).