Rat Transient Receptor Potential Vanilloid 1 Research Articles

Hypoxia-induced sensitization of transient receptor potential vanilloid 1 involves activation of hypoxia-inducible factor-1 alpha and PKC

The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), acts as a polymodal detector of pain-producing chemical and physical stimuli in sensory neurons. Hyperglycemia and hypoxia are two main phenomena in diabetes associated with several complications. Although many studies on streptozotocin-induced diabetic rats indicate that early diabetic neuropathy is associated with potentiation of TRPV1 activity in dorsal root ganglion neurons, its underlying mechanism and distinctive roles of hyperglycemia and hypoxia have not been completely clarified. Here, we show that hypoxic and high glucose conditions (overnight exposure) potentiate the TRPV1 activity without affecting TRPV1 expression in both native rat sensory neurons and human embryonic kidney-derived 293 cells expressing rat or human TRPV1. Surprisingly, hypoxia was found to be a more effective determinant than high glucose, and hypoxia-inducible factor-1 alpha (HIF-1α) seemed to be involved. In addition, high glucose enhanced TRPV1 sensitization only when high glucose existed together with hypoxia. The potentiation of TRPV1 was caused by its phosphorylation of the serine residues, and translocation of protein kinase C (PKC)ε was clearly observed in the cells exposed to the hypoxic conditions in both cell types, which was inhibited by 2-methoxyestradiol, a HIF-1α inhibitor. These data suggest that hypoxia is a new sensitization mechanism for TRPV1, which might be relevant to diabetes-related complications, and also for other diseases that are associated with acute hypoxia.TRPV1 was found to be sensitized upon in vitro overnight exposure to hypoxia and high glucose mainly by hypoxia in a PKCepsilon- and HIF-1alpha-dependent manner.

Rat 바닐로이드 수용체 TRPV1과 Rab11-FIP3의 특이적 결합

캡사이신 채널로 알려진 바닐로이드 수용체 TRPV1 (캡사이신채널, Transient Receptor Potential Vanilloid 1)은 통증발현에서 중요한 역할을 하는 것으로 알려져 있다. 하지만 TRPV1의 활성조절에 관여하는 단백질에 대하여는 알려진 바가 많지 않다. 최근 rat TRPV1과 직접적으로 결합하는 단백질을 탐색하여 mouse Rab11-FIP3 (rab11-family interaction protein 3)가 rat TRPV1과 직접적으로 결합한다는 것이 보고되었다. Rab11은 여러 가지의 세포내 이동에 관여하는 것으로 보고되었다. 그러므로 Rab11-FIP3과의 결합을 통해 TRPV1의 세포막으로의 이동에 관여할 것으로 추측할 수 있다. 본 연구에서는 전에 보고된 연구가 mouse와 rat 이라는 다른 종의 단백질끼리의 결합이기 때문에 같은 종에서의 상호작용을 확인하고 Rab11-FIP3의 TRPV1의 세포막으로의 이동에서의 역할을 알아보고자 현재까지 동정되지 않은 rat의 Rab11-FIP3의 유전자를 GenBank 서열을 바탕으로 rat 뇌의 RNA 로부터 cDNA 를 클로닝하여 유전자를 분리하고 TRPV1 과의 관계를 세포생물학적으로 알아보았다. 연구결과 rat의 Rab11-FIP3는 489개의 아미노산 서열을 가지고 있으며 human과는 80%, mouse와는 90% 이상 아미노산 서열의 상동성을 보였다. 조직별 분포는 심장, 뇌, 간, 콩팥, 정소에서 발현되고 있는 것을 northern blot assay와 western blot assay 로 확인하였다. rat 의 뇌조직에서 TRPV1 과 Rab11-FIP3 단백질이 결합하여 colocalize 하는 것을 면역화학방법으로 확인하였다. 이 결합은 같은 family 의 TRPV2 와는 결합하지 않는 특이적 결합이므로 Rab11-FIP3 가 TRPV1 과 상호작용하여 세포막으로의 이동에 관여할 것이라는 것을 시사한다. Vanilloid receptor TRPV1 (known as capsaicin channel, transient receptor potential vanilloid 1) is known to be a key protein in the pain signal transduction. However, the proteins controlling the activity of the channel are not much known yet. Recently mouse Rab11-FIP3 (Rab11-family interaction protein 3) was found and reported to interact with rat TRPV1. Rab11 has been shown to play a key role in a variety of cellular processes including plasma membrane recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. Therefore, Rab11-FIP3 was proposed to be involved in the membrane trafficking of TRPV1. In this study, the unreported rat Rab11-FIP3 was yet cloned in order to show the specific interaction of the TRPV1 and Rab11-FIP3 in the same species of rat and to examine the membrane trafficking of TRPV1. The result showed that rat Rab11-FIP3 is expected to have 489 amino acids and showed 80% identity with that of human and over 90% identity with that of mouse. Rab11-FIP3 was found to be expressed in heart, brain, kidney, testis using northern and western blot analyses. We also found that rat Rab11-FIP3 was colocalized with rat TRPV1 but not with TRPV2 of same family in the rat brain by using immunohistochemistry showing that two proteins interact specifically, suggesting the role of Rab11-FIP3 in the membrane trafficking.

Anesthesia Literature Review

Anesthesia Literature Review

17-Beta-estradiol enhanced allodynia of inflammatory temporomandibular joint through upregulation of hippocampal TRPV1 in ovariectomized rats.

Temporomandibular disorders (TMDs) predominantly affect reproductive female patients, with pain the most frequent complaint. Although estrogens are believed to play important roles in TMD pain, the mechanism underlying modulation of TMD pain by estrogens remains largely unknown. Accumulating evidence implies that the hippocampus is involved in sexual dimorphism of pain sensitivity. In this study, we investigated the hippocampal TRPV1 (transient receptor potential vanilloid 1) expression in ovariectomized rats that received 17-beta-estradiol substitution and found that 17-beta-estradiol enhanced the mechanical allodynia of inflamed temporomandibular joint (TMJ) induced by complete Freund's adjuvant. Real-time PCR and immunoblotting demonstrated that TMJ inflammation significantly induced hippocampal TRPV1 expression compared with the control group but failed to induce it in the ovariectomized rats that received no estradiol replacement. In addition, estradiol potentiated TMJ inflammation-induced hippocampal TRPV1 expression in a dose-dependent manner in the ovariectomized rats. In contrast, TRPV1 transcription in amygdala, prefrontal cortex, and thalamus was not affected by TMJ inflammation and estradiol. Immunostaining showed TRPV1 localized in the processes and cytoplasm of pyramidal neurons in CA1-CA3 regions of the hippocampus. Moreover, intrahippocampal injection of TRPV1 antagonists capsazepine and 5'-iodo-resiniferatoxin into the CA1 region of the hippocampus significantly attenuated allodynia of inflamed TMJ in both nonovariectomized and ovariectomized rats that received estradiol replacement. Our results suggested that hippocampal TRPV1 can modulate central pain processing and estradiol may contribute to the sexual dimorphism of TMD pain sensitivity through upregulation of TRPV1 expression in the hippocampus.

Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents

The transient receptor potential vanilloid 1 (TRPV1) channel is the principal detector of noxious heat in the peripheral nervous system. TRPV1 is expressed in many nociceptors and is involved in heat-induced hyperalgesia and thermoregulation. The precise mechanism or mechanisms mediating the thermal sensitivity of TRPV1 are unknown. Here, we have shown that the oxidized linoleic acid metabolites 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE) are formed in mouse and rat skin biopsies by exposure to noxious heat. 9- and 13-HODE and their metabolites, 9- and 13-oxoODE, activated TRPV1 and therefore constitute a family of endogenous TRPV1 agonists. Moreover, blocking these substances substantially decreased the heat sensitivity of TRPV1 in rats and mice and reduced nociception. Collectively, our results indicate that HODEs contribute to the heat sensitivity of TRPV1 in rodents. Because oxidized linoleic acid metabolites are released during cell injury, these findings suggest a mechanism for integrating the hyperalgesic and proinflammatory roles of TRPV1 and linoleic acid metabolites and may provide the foundation for investigating new classes of analgesic drugs.

Binding of [ 3H]A-778317 to native transient receptor potential vanilloid-1 (TRPV1) channels in rat dorsal root ganglia and spinal cord

A-778317 (1-((R)-5- tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea) is a potent antagonist of human and rat transient receptor potential vanilloid-1 (TRPV1) receptors. We have previously reported that [ 3H]A-778317 is an excellent radioligand to study the recombinant human TRPV1 receptor in a heterologous expression system. These studies were extended to determine the feasibility of using [ 3H]A-778317 to label native TRPV1 channels in rat tissues. Saturable high-affinity binding of [ 3H]A-778317 was detected in membrane preparations of rat dorsal root ganglia (DRG) and spinal cord that was inhibited by TRPV1 receptor agonists and antagonists. [ 3H]A-778317 labeled a single class of high-affinity binding sites in both rat DRG and spinal cord membranes ( K D = 10 and 8.4 nM, respectively). The number of binding sites was 10-fold higher in rat DRG membranes than spinal cord membranes ( B max = 3.3 and 0.35 pmol/mg protein, respectively). The pharmacology of the high-affinity binding sites was similar in rat DRG and spinal cord, but differed from the recombinant rat TRPV1 (rTRPV1) receptor expressed in transiently transfected HEK293-F cells. In particular, a large disparity in potency (> 300-fold) was observed for the TRPV1 receptor agonist resiniferatoxin between native and recombinant rTRPV1 receptors. Our data indicate that the binding of [ 3H]A-778317 to native rTRPV1 channels is pharmacologically distinct, and perhaps more complex, than its binding to the recombinant rTRPV1 receptor.

Gosha‐jinki‐gan reduces transmitter proteins and sensory receptors associated with C fiber activation induced by acetic acid in rat urinary bladder

We determined if Gosha-jinki-gan, a traditional Chinese herbal mixture, reduced the presence of the tachykinins neurokinin A, neurokinin B, and substance P, as well as the transient receptor potential vanilloid 1 (TRPV1) and P2X3 purine receptors that are functionally associated with C fibers in the urinary bladder. Thirty-six female rats were fed with either a standard diet or one supplemented with 1.08% Gosha-jinki-gan. After 4 weeks, the urinary bladders were instilled with either saline or 0.1% acetic acid. After 30 min, the bladders were removed and expression of the tachykinins and the TRPV1 and P2X3 receptors was determined by immunohistochemistry and mRNA expression. In rats fed with the standard diet, the tachykinins and the TRPV1 and P2X3 receptors expressed nearby or within urothelium of the acetic acid-treated rats increased compared with the saline-instilled rats. In rats pretreated with Gosha-jinki-gan, the tachykinins and the TRPV1 and P2X3 receptors in the acetic acid-treated rats also increased compared with the saline-instilled rats. However, with the instillation of acetic acid, the tachykinins and the TRPV1 and P2X3 receptors of Gosha-jinki-gan pretreated rats decreased compared with standard diet fed rats. The mRNA expression levels of neurokinin A, substance P, and the TRPV1 receptor in acetic acid-treated Gosha-jinki-gan pretreated rats were lower than that in acetic acid-treated standard diet fed rats. Gosha-jinki-gan did not destroy nerve fibers within the bladders. Gosha-jinki-gan partially reduced the tachykinins and TRPV1 and P2X3 purine receptors without destroying the nerve fibers.

TRANSIENT RECEPTOR POTENTIAL VANILLOID-1–MEDIATED CALCIUM RESPONSES ARE INHIBITED BY THE ALKYLAMINE ANTIHISTAMINES DEXBROMPHENIRAMINE AND CHLORPHENIRAMINE

American guidelines, unlike European guidelines, support the use of antihistamines as a first line of treatment for some causes of chronic cough. Transient receptor potential vanilloid-1 (TRPV1) is an ion channel activated by the tussive agents capsaicin, resiniferatoxin, and protons. It is predominantly expressed by C-fiber and some Aδ -fiber sensory neurons and is thought to be a cough receptor. By measuring increases in intracellular calcium as an indicator of TRPV1 activation, the authors sought to determine whether antihistamines could antagonise TRPV1 permanently expressed in HEK and Pro5 cells and TRPV1 endogenously expressed in rat dorsal root ganglia neurons. In human TRPV1–expressing HEK cells (hTRPV1-HEK), diphenhydramine and fexofenadine failed to inhibit capsaicin-triggered calcium responses. However, both dexbrompheniramine and chlorpheniramine significantly inhibited capsaicin-evoked responses in hTRPV1-HEK. Dexbrompheniramine also inhibited activation of rat TRPV1 expressed in HEK and Pro5 cells, without interfering with TRPA1 and proteinase-activated receptor-2 (PAR2) activation. Finally, in rat dorsal root ganglia neuron preparations, dexbrompheniramine dose-dependently inhibited capsaicin-evoked calcium responses. Thus, the inhibition of TRPV1 activation by dexbrompheniramine may provide one potential mechanism whereby this antihistamine exerts its therapeutic effect in chronic cough.

3H]A-778317 [1-((R)-5-tert-Butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: a Novel, Stereoselective, High-Affinity Antagonist Is a Useful Radioligand for the Human Transient Receptor Potential Vanilloid-1 (TRPV1) Receptor

1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 +/- 0.13). The (S)-stereoisomer, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778316), is 6.8-fold less potent (pIC50 = 7.47 +/- 0.07). A-778317 also potently blocks capsaicin and acid activation of native rat TRPV1 receptors in dorsal root ganglion neurons. A-778317 was tritiated ([3H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [3H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (KD = 3.4 nM; Bmax = 4.0 pmol/mg protein). Specific binding of 2 nM [3H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [3H]A-778317 correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A-778317 blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site and that [3H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology.

Analgesic actions of N‐arachidonoyl‐serotonin, a fatty acid amide hydrolase inhibitor with antagonistic activity at vanilloid TRPV1 receptors

N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia. AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured. AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM251, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone. Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.

Identification and functional study of an alternatively spliced variant of rat TRPV1 receptor mRNA

Transient receptor potential vanilloid 1(TRPV1), or Vanilloid receptor 1(VR1), is a member of the TRP superfamily of nonselective cation channels. TRPV1 consists of a six transmembrane-spanning domain, a pore region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. This receptor is activated by several endogenous ligands and noxious heat, acid as well as inflammatory factors, and has been proposed as a potential target for the treatment of pain and inflammation. Although numerous studies have reported the characterization of the splicing variants of rat, mouse and human TPRV1, these mRNA variants encoded N-terminal deletion forms of TRPV1 receptors that are inactiveisoforms of TRPV1 receptors. Here, we report the identification and molecular cloning of a C-terminal truncated form of rat TRPV1 receptor isolated from rat stomach and small intestines cDNA libraries. The deduced protein is designated as TRPV1c and contains 760 amino acids. When TRPV1c was expressed in HEK 293 cells, the TRPV1c acted as a ligand-gated, Ca2+ permeable channel with similar agonist and antagonist pharmacology to wild type rat TRPV1 in FLIPR-based Ca2+ assays. The ligands- stimulated TRPV1c activation was also shown to be dependent on the assay pH. Higher (pH 9) or lower (pH 5.5) assay pH will dramatically reduce ligands-stimulated TRPV1c activity.

Activation of transient receptor potential vanilloid 1 (TRPV1) by resiniferatoxin

Transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+) permeable non-selective cation channel activated by physical and chemical stimuli. Resiniferatoxin (RTX), an ultrapotent agonist of TRPV1, is under investigation for treatment of urinary bladder hyper-reflexia and chronic pain conditions. Here, we have determined the characteristics of RTX-induced responses in cells expressing native and cloned rat TRPV1. Whole-cell currents increase with repeated application of submaximal concentrations of RTX until a maximal response is attained and do not deactivate even after prolonged washout. Interestingly, the rate of activation and block by capsazepine of RTX-induced currents are significantly slower than for capsaicin-induced currents. RTX-induced whole-cell currents are outwardly rectifying, but to a lesser extent than capsaicin-induced currents. RTX-induced single channel currents exhibit multiple conductance states and outward rectification. The open probability (P(o)) of RTX-induced currents is higher at all potentials as compared to capsaicin-induced currents, which showed a strong voltage-dependent decrease at negative potentials. Single-channel kinetic analyses reveal that open-time distribution of RTX-induced currents can be fitted with three exponential components at negative and positive potentials. The areas of distribution of the longer open time constants are significantly larger than capsaicin-induced currents. The closed-time distribution of RTX-induced currents can be fitted with three exponential components as compared to capsaicin-induced currents, which require four exponential components. Current-clamp experiments reveal that low concentrations of RTX caused a slow and sustained depolarization beyond threshold while generating few action potentials. Concentrations of capsaicin required for the same extent of depolarization generated a significantly greater number of action potentials. These properties of RTX may play a role in its clinical usefulness.

5-Iodoresiniferatoxin Evokes Hypothermia in Mice and Is a Partial Transient Receptor Potential Vanilloid 1 Agonist in Vitro

Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated ion channel required for normal in vivo responses to these painful stimuli. However, growing evidence suggests that TRPV1 also participates in thermoregulation. Therefore, we examined the effects of a selective TRPV1 antagonist, 5-iodoresiniferatoxin (I-RTX), on mouse body temperature. Surprisingly, s.c. administration of I-RTX (0.1-1 micromol/kg) evoked a hypothermic response similar to that evoked by capsaicin (9.8 micromol/kg) in naive wild-type mice, but not in mice pretreated with resiniferatoxin, a potent TRPV1 agonist, or in naive TRPV1-null mice. In response to I-RTX in vitro, HEK293 cells expressing rat TRPV1 exhibited increases in intracellular Ca(2+) (biphasic, EC(50) = 56.7 nM and 9.9 microM) that depended on Ca(2+) influx and outwardly rectifying, capsazepine-sensitive currents that were smaller than those evoked by 1 microM capsaicin. Thus, I-RTX induces TRPV1-dependent hypothermia in vivo and is a partial TRPV1 agonist in vitro.

An Alternative Splicing Product of the Murine trpv1 Gene Dominant Negatively Modulates the Activity of TRPV1 Channels

Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1alpha and TRPV1beta and contain 839 and 829 amino acids, respectively. TRPV1beta arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1alpha in many tissues and significant expression of TRPV1beta in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1alpha formed a Ca(2+)-permeable channel activated by ligands known to stimulate TRPV1. TRPV1beta was not functional by itself but its co-expression inhibited the function of TRPV1alpha. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1beta than TRPV1alpha protein was found in cells and on the cell surface, indicating that the beta isoform is highly unstable. Our data suggest that TRPV1beta is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli.