Transient Receptor Potential V1 Research Articles

Eosinophils Increase Transient Receptor Potential V1 and Substance P Expression in Dorsal Root Ganglia In Vivo

Background: Asthma is characterized by excessive bronchoconstriction and cough. Airway nerves control these reflexes. In asthma, an abundance of airway eosinophils cause airway nerve dysfunction by altering neurotransmitter content and neuronal receptor expression.

Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.

Psoriasis is characterized by keratinocyte hyperproliferation, erythema, as well as a form of pruritus, involving cutaneous discomfort. There is evidence from both clinical and murine models of psoriasis that chemical or surgical depletion of small-diameter sensory nerves/nociceptors benefits the condition, but the mechanisms are unclear. Hence, we aimed to understand the involvement of sensory nerve mediators with a murine model of psoriasis and associated spontaneous behaviors, indicative of cutaneous discomfort. We have established an Aldara model of psoriasis in mice and chemically depleted the small-diameter nociceptors in a selective manner. The spontaneous behaviors, in addition to the erythema and skin pathology, were markedly improved. Attenuated inflammation was associated with reduced dermal macrophage influx and production of reactive oxygen/nitrogen species (peroxynitrite and protein nitrosylation). Subsequently, this directly influenced observed behavioral responses. However, the blockade of common sensory neurogenic mechanisms for transient receptor potential (TRP)V1, TRPA1, and neuropeptides (substance P and calcitonin gene-related peptide) using genetic and pharmacological approaches inhibited the behaviors but not the inflammation. Thus, a critical role of the established sensory TRP–neuropeptide pathway in influencing cutaneous discomfort is revealed, indicating the therapeutic potential of agents that block that pathway. The ongoing inflammation is mediated by a distinct sensory pathway involving macrophage activation.—Kodji, X., Arkless, K. L., Kee, Z., Cleary, S. J., Aubdool, A. A., Evans, E., Caton, P., Pitchford, S. C., Brain, S. D. Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.

Abstract 084: Selective Renal Afferent Denervation Enhances Diuretic and Natriuretic Response to Glucagon-like Peptide-1 in Rats With Heart Failure

Glucagon-like peptide-1 (GLP-1), incretin hormone, has diuretic and natriuretic effects and its receptor agonist reduces cardiovascular events. The present study was designed to determine whether GLP-1-induced diuresis is regulated by renal nerve in the rats with a coronary ligation model of heart failure (HF). Our immunohistochemistry and immunoblotting studies demonstrate that GLP-1 receptors were expressed in the renal pelvic wall, densely innervated by afferent renal nerves (ARN). Basal ARN activity (ARNA) was increased in HF compared to normal rats (1.3 ± 0.2* vs. 0.6 ± 0.1 μV·s). GLP-1 (3μM) infused into left renal pelvis increased ipsilateral ARNA in both normal and HF rats. The change in basal ARNA and the maximum ARNA represented as percent activation by injection of capsaicin (transient receptor potential V1 receptor agonist, 100μM) were higher in HF than in normal rats (ΔARNA 227 ± 27%* vs. 131 ± 26%, and ARNAmax 64.2 ± 3.4%* vs. 45.1 ± 6.6%). Intravenous infusion of GLP-1 (1μg/kg/min) for 30 min increased renal urine flow and sodium excretion in normal rats and these effects were blunted in HF (Urine flow 1577 ± 122* vs. 1981 ± 101 μl/gkw, Sodium excretion 221 ± 19* vs. 284 ± 15 μEq/gkw). GLP-1-induced diuresis and natriuresis were enhanced by total renal denervation (T-RDN) with acute surgical cutting of the renal nerves in both normal and HF rats. The degree of diuretic and natriuretic responses to GLP-1 were greater in HF than in normal rats (increased % rate: Urine flow 167%* vs. 135%, Sodium excretion 169%* vs. 150%). Selective afferent renal denervation (A-RDN) was performed by bilateral perivascular application of capsaicin (33mM) on the renal nerves. Ten days after A-RDN, there was an enhanced diuretic and natriuretic response to GLP-1 in both normal and HF rats. These effects were greater in HF than in normal rats (increased % rate: Urine flow 187%* vs. 130%, Sodium excretion 147%* vs. 138%). Urine flow and sodium excretion responses to GLP-1 were not significantly different between T-RDN and A-RDN in both groups. Taken together our results indicate that the diuretic and natriuretic effects of GLP-1 are partly governed via ARN activation. GLP-1-induced diuresis and natriuresis enhanced by RDN have potential therapeutic implication for HF.

Potentiation of capsaicin-induced neurogenic inflammation by 5-HT7 receptors in the rat hind paw: Involvement of calcitonin gen-related peptide

A decrease in the activation threshold of primary sensory neurons to transient receptor potential V1 (TRPV1) stimulation by serotonin 5-HT7 receptors has been reported but no confirmation if this might translate into facilitation of neurogenic inflammation has been provided. We analysed the modulation of capsaicin (CAP)-induced neurogenic inflammation in the rat hind paw by the selective 5-HT7 receptor agonist, LP-44, and the involvement of calcitonin gen-related peptide (CGRP) in this effect. Animals received intra-plantar injections (30 μL) of vehicle, CAP (0.05%, 0.1% and 0.2%), LP-44 (7.5 and 15 nmol) and the combination of LP–44 + CAP; then, the time course of the inflammatory responses was measured. The effect of the 5-HT7 receptor antagonist, SB-269970 (3 mg/kg, s.c.), on responses produced by LP-44 alone and combined with CAP was tested. As expected, CAP produced concentration- and time-dependent inflammatory responses in the hind paw. Interestingly, LP-44 by itself also produced inflammation in a concentration- and time-dependent manner, and magnified CAP-induced responses. Systemic pre-treatment with SB-269970 significantly blunted LP-44 (15 nmol)-induced inflammation as well as magnified inflammatory responses produced by the combination of LP-44 (7.5 and 15 nmol) + CAP (0.1%) thus confirming the involvement of 5-HT7 receptors. Finally, the non-peptide CGRP receptor antagonist, BIBN4096 (3 mg/kg, s.c.), strongly inhibited the potentiated inflammatory responses induced by LP-44 (7.5 and 15 nmol) + CAP (0.1%) thus substantiating their neurogenic nature. Thus, sensitization of CAP-sensitive primary sensory neurons by 5-HT7 receptors may result in facilitation of neurogenic inflammation involving CGRP in the rat hind paw.

Allyl isothiocyanate increases carbohydrate oxidation through enhancing insulin secretion by TRPV1.

The transient receptor potential (TRP) V1 is a cation channel belonging to the TRP channel family and it has been reported to be involved in energy metabolism, especially glucose metabolism. While, we have previously shown that intragastric administration of allyl isothiocyanate (AITC) enhanced glucose metabolism via TRPV1, the underlying mechanism has not been elucidated. In this study, we examined the relationship between insulin secretion and the increase in carbohydrate oxidation due to AITC. Intragastric administration of AITC elevated blood insulin levels in mice and AITC directly enhanced insulin secretion from isolated islets. These observations were not reproduced in TRPV1 knockout mice. Furthermore, AITC did not increase carbohydrate oxidation in streptozotocin-treated mice. These results suggest that intragastric administration of AITC could induce insulin secretion from islets via TRPV1 and that enhancement of insulin secretion was related to the increased carbohydrate oxidation due to AITC.

Hyperglycemia‐Induced Alteration of Brain Microvascular Endothelial Intracellular Ca Response to Ischemic Factors: Role of TRPV4 Channels

Cerebral edema is exacerbated in hyperglycemic ischemic stroke through poorly understood mechanisms. We showed previously that hypoxia and other factors present during the early hours of ischemic stroke stimulate blood‐brain barrier (BBB) Na‐K‐Cl cotransport (NKCC1) and Na/H exchange (NHE1) activities, leading to increased secretion of Na, Cl and water from blood into brain across a still intact BBB and consequent edema formation. More recently we found that BBB endothelial cells exposed to high glucose (6 hr to 7 d) exhibit increased abundance of NKCC and NHE and greater stimulation of the transporter activities by ischemic factors compared to cells maintained in normoglycemic conditions. Further, we found that the more robust edema formation observed in hyperglycemic rats is significantly attenuated by bumetanide and HOE‐642, inhibitors of NKCC1 and NHE1 activities. The present study was conducted to investigate signaling events underlying these effects of hyperglycemia on BBB Na transporters and ischemia‐induced cerebral edema with the overall goal of identifying additional therapeutic targets for diabetic ischemic stroke. Compelling evidence suggests that intracellular Ca (Cai) signaling mechanisms are likely to be involved. Exposure to high glucose is known to alter Cai dynamics in several cell types, including non‐brain endothelial cells. In addition, stimulation of NKCC and/or NHE activity by increases in Cai has been demonstrated in a variety of cell types. In recent studies we found that exposing BBB endothelial cells to high glucose for 48 hours resulted in elevated resting levels of Cai and significantly increased the abundance of the Transient Receptor Potential V4 (TRPV4) channel. The present study was conducted to further evaluate the effects of hyperglycemia on BBB endothelial cell Cai and to investigate whether TRPV4 plays a role in hyperglycemia‐induced alteration of Cai dynamics in the cells. Bovine cerebral microvascular endothelial cells (CMEC) were exposed for 24 hr to hyperglycemic (HG, 30mM glucose), normoglycemic (NG, 5mM glucose) or osmotic control media (5mM glucose + 12.5 mM NaCl) then loaded with 5μM Cal‐520‐AM and imaged after exposure (1 hr) to normoxia or hypoxia (7% O2). Calcium levels were normalized to maximal non‐saturated calcium signal following the addition of 8μM ionomycin. We found that HG CMEC exhibited a 4‐fold increase in [Cai] when exposed to hypoxia compared to a 2‐fold in NG CMEC. This effect was abolished in the presence Ca‐free media or the TRPV4 inhibitor HC067047 (1μM). Western blot evaluation of CMEC lysates following exposure to HG media (6 to 48 hr) revealed significant increases in TRPV4 abundance after 24 and 48 hr but no effect of osmotic control medium (NG media with added 12.5 mM NaCl). Using conventional perforated patch‐clamp methods and a 1‐second voltage ramp (−100 mV to +100 mV) protocol, we also found that the TRPV4 agonist GSK1016790A activated an outwardly rectifying current in the cells that was blocked by the antagonist (HC067047; 1μM), indicating the presence of TRPV4 activity in the cells. Our findings indicate that hyperglycemia exposures cause more robust hypoxia‐induced increases in BBB endothelial cell [Cai] in a TRPV4‐dependent manner.Support or Funding InformationSupported by NINDS and the American Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial ROS activates PKC alpha translocation to the membrane of vagal sensory neurons.

Hyperexcitability of airway sensory nerves can trigger disease‐associated reflexes resulting in cough and dyspnea. We have observed that mitochondrial ROS production increases hyperexcitability in nociceptive bronchopulmonary C‐fibers. We also found that this increased excitability can be blocked by the PKC inhibitor bisindolylmaleimide (BIM) I, but not the inactive PKC analog BIM V. In previous work we have shown, using a pan‐PKC antibody, that mitochondrial ROS production via Antimycin A (AntA; mitochondrial complex III Qi site inhibitor) causes translocation to the cell membrane. However, there are several PKC isoforms expressed in nociceptive neurons. We wanted to determine which isoforms were present, their distribution in nociceptive cells and whether specific isoforms were activated by AntA. Studies looking at mRNA expression have highlighted PKC alpha, beta1, beta2, delta and epsilon as abundant in nociceptors so our studies have focused on these particular isoforms. Sensory neuron cell bodies were isolated from mouse vagal ganglia. To identify nociceptive cells, we used a mouse strain that co‐expresses a red fluorescent protein (tdTomato) with the Transient Receptor Potential V1 ion channel. Cells were treated for 2 min with: vehicle, 10 μM AntA or 300 nM PMA then immediately fixed. We performed immunocytochemistry to determine cellular location of different isoforms. Of the TRPV1 expressing nociceptive cells, more than 50% were positive for any given isoform of PKC. All isoforms translocated to the membrane in response to PMA treatment. However, only PKC alpha exhibited translocation to the membrane in response to AntA treatment. In the case of PKC delta, AntA appeared to cause translocation of this isoform to the nucleus. Our experiments suggest that nociceptor hyperexcitability, in response to ROS produced via mitochondrial dysfunction, may occur through the translocation and activation of the PKC alpha isoform.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Association of TRPV1 and TLR4 through the TIR domain potentiates TRPV1 activity by blocking activation-induced desensitization.

BackgroundWe have previously reported that histamine-induced pruritus was attenuated in toll-like receptor 4 (TLR4) knockout mice due to decreased transient receptor potential V1 (TRPV1) sensitivity. Our results implied that TLR4 potentiated TRPV1 activation in sensory neurons; however, the molecular mechanism has yet to be elucidated. In this study, we investigated the molecular mechanisms of TLR4-mediated TRPV1 potentiation using TLR4-deficient sensory neurons and a heterologous expression system.MethodsPrimary sensory neurons were obtained from wild-type or TLR4 knockout mice, and HEK293T cells expressing TRPV1 and TLR4 were prepared by transient transfection. TRPV1 activity was analyzed by calcium imaging, fluorophotometry, and patch-clamp recording. Subcellular protein distribution was tested by immunocytochemistry and cell surface biotinylation assay. Protein interaction was assessed by western blot and immunoprecipitation assay.ResultsDirect association between TRPV1 and TLR4 was detected in HEK293T cells upon heterologous TRPV1 and TLR4 expression. In an immunoprecipitation assay using TLR4-deletion mutants and soluble toll/interleukin-1 receptor (TIR) protein, the cytoplasmic TIR domain of TLR4 was required for TLR4-TRPV1 association and TRPV1 potentiation. In TLR4-deficient sensory neurons, the activation-induced desensitization of TRPV1 increased, accompanied by enhanced TRPV1 clearance from the cell membrane upon activation compared to wild-type neurons. In addition, heterologous TLR4 expression inhibited activation-induced TRPV1 endocytosis and lysosomal degradation in HEK293T cells.ConclusionOur data show that direct association between TRPV1 and TLR4 through the TIR domain enhances TRPV1 activity by blocking activation-induced TRPV1 desensitization.

Dyslipidemia modulates Müller glial sensing and transduction of ambient information.

Unesterified cholesterol controls the fluidity, permeability and electrical properties of eukaryotic cell membranes. Consequently, cholesterol levels in the retina and the brain are tightly regulated whereas depletion or oversupply caused by diet or heredity contribute to neurodegenerative diseases and vision loss. Astroglia play a central role in the biosynthesis, uptake and transport of cholesterol and also drive inflammatory signaling under hypercholesterolemic conditions associated with high-fat diet (diabetes) and neurodegenerative disease. A growing body of evidence shows that unesterified membrane cholesterol modulates the ability of glia to sense and transduce ambient information. Cholesterol-dependence of Müller glia - which function as retinal sentinels for metabolic, mechanical, osmotic and inflammatory signals - is mediated in part by transient receptor potential V4 (TRPV4) channels. Cholesterol supplementation facilitates, whereas depletion suppresses, TRPV4-mediated transduction of temperature and lipid agonists in Müller cells. Acute effects of cholesterol supplementation/depletion on plasma membrane ion channels and calcium homeostasis differ markedly from the effects of chronic dyslipidemia, possibly due to differential modulation of modality-dependent energy barriers associated with the functionality of polymodal channels embedded within lipid rafts. Understanding of cholesterol-dependence of TRP channels is thus providing insight into dyslipidemic pathologies associated with diabetic retinopathy, glaucoma and macular degeneration.

Perilla extract improves frequent urination in spontaneously hypertensive rats with enhancement of the urothelial presence and anti-inflammatory effects.

To investigate the effects of perilla extract on urinary symptoms in spontaneously hypertensive rats as a model of spontaneous overactive bladder. Spontaneously hypertensive rats were randomly divided into two groups and fed either a control diet or a perilla extract-containing diet. Cystometry, gene expression and histological analyses were carried out to evaluate the effects of perilla extract after 2-week feeding of either the control or the perilla extract diet. The expression of inflammation-related genes in the human urothelial cell line HT-1376 and the normal human bladder epithelial cell was measured after the treatment with perillaldehyde, the main component of perilla extract, or perillic acid, the final metabolite of perillaldehyde. A significant 27% increase in the micturition interval and decreased expression of nerve growth factor, tumor necrosis factor-α, interleukin-1β and transient receptor potential V1 were observed in the perilla group compared with the control group. The level of uroplakin 3A was 40% higher in the perilla group than in the control group. The urothelium in the control group was thin or defective, but it was almost completely intact in the perilla group. Perillaldehyde and perillic acid suppressed the induction of nerve growth factor and tumor necrosis factor-α by interleukin-1β in HT-1376 and normal human bladder epithelial cells. The present findings suggest that perilla extract improves frequent urination, and this improvement seems to be mediated, at least in part, by enhancement of the urothelial presence and by the anti-inflammatory effects of perilla.

Abstract P503: Role of TRPV4 and Endothelial BK Channels in Endothelial-dependent Dilation Following Chronic Hypoxia

Following chronic hypoxia (CH) the systemic vasculature exhibits blunted vasoconstriction due to endothelial cell-dependent hyperpolarization (EDH). Previous data demonstrate that after CH, EDH-mediated dilation switches from a reliance on SK ca and IK ca channels to activation of endothelial BK ca channel (eBK). The mechanism by which endothelial cell stimulation activates eBK channels following CH is not known. We hypothesized that following CH, EDH-dependent dilation involves a transient receptor potential V4 (TRPV4)-dependent activation of eBK channels. ACh induced concentration-dependent dilation in pressurized gracilis arteries from normoxic and CH rats. TRPV4 blockade only attenuated ACh-induced vasodilation in arteries from CH rats (Fig. 1). ACh elicited an increase in endothelial TRPV4-dependent Ca 2+ events in arteries from both groups. Direct activation of TRPV4 elicited dilation in arteries from normoxic and CH rats (97 ± 2 % and 95 ± 2 % for normoxic and CH, respectively). In arteries from normoxic rats, inhibition of SK ca and IK ca abolished dilation to TRPV4 activation (6 ± 5 %), whereas, luminal iberiotoxin had no effect (89 ± 7 %). In CH rats, only administration of all three K ca channel inhibitors abolished dilation to TRPV4 activation (3 ± 10%). Disruption of endothelial caveolae with methyl-β-cyclodextrin significantly decreased ACh-induced dilation in arteries from both groups. Using a proximity ligation assay, we observed co-localization between caveolin-1, TRPV4, and eBK channels in the endothelium of gracilis arteries. In conclusion, CH results in functional coupling between muscarinic receptors, TRPV4 and K ca channels in gracilis arteries.

TRPV1 receptor-mediated expression of Toll-like receptors 2 and 4 following permanent middle cerebral artery occlusion in rats.

Objective(s):Stroke is known as a main cause of mortality and prolonged disability in adults. Both transient receptor potential V1 (TRPV1) channels and toll-like receptors (TLRs) are involved in mediating the inflammatory responses. In the present study, the effects of TRPV1 receptor activation and blockade on stroke outcome and gene expression of TLR2 and TLR4 were assessed following permanent middle cerebral artery occlusion in ratsMaterials and Methods:Eighty male Wistar rats were divided into four groups as follows: sham, vehicle, AMG9810 (TRPV1 antagonist) -treated and capsaicin (TRPV1 agonist) -treated. For Stroke induction, the middle cerebral artery was permanently occluded and then behavioral functions were evaluated 1, 3 and 7 days after stroke.Results:TRPV1 antagonism significantly reduced the infarct volume compared to the stroke group. Also, neurological deficits were decreased by AMG9810 seven days after cerebral ischemia. In the ledged beam-walking test, the slip ratio was enhanced following ischemia. AMG9810 decreased this index in stroke animals. However, capsaicin improved the ratio 3 and 7 days after cerebral ischemia. Compared to the sham group, the mRNA expression of TLR2 and TLR4 was significantly increased in the stroke rats. AMG9810 Administration significantly reduced the mRNA expression of TLR2 and TLR4. However, capsaicin did not significantly affect the gene expression of TLR2 and TLR4.Conclusion:Our results demonstrated that TRPV1 antagonism by AMG9810 attenuates behavioral function and mRNA expression of TLR2 and TLR4. Thus, it might be useful to shed light on future therapeutic strategies for the treatment of ischemic stroke.

Transient receptor potential a1 (TRPA1) agonists inhibit contractions of the isolated human ureter.

Mechanoafferent and peristaltic mechanisms of the human ureter involve transient receptor potential V1 (TRPV1)- and purinoceptor-mediated functions. Hydrogen sulphide, an endogenous TRPA1 ligand, is linked to inhibitory neurotransmission of the pig ureter. No information is available on TRPA1 activity in the human ureter. We therefore examined the distribution and function of TRPA1 in the human ureter. Expression of TRPA1 in human ureter tissue was studied by Western blot and immunofluorescence. The TRPA1 distribution was compared to TRPV1, calcitonin gene related peptide (CGRP), tyrosine hydroxylase (TH), and vimentin. Effects of the TRPA1 agonists allyl isothiocyanate (AI), cinnamaldehyde (CA), sodium hydrogen sulfide (NaHS), and capsaicin (TRPV1 agonist) on human ureter preparations were studied in organ baths. By Western blot, bands were detected at the expected molecular weight for TRPA1. TRPA1- and TRPV1-immunoreactivities were located on CGRP-positive nerves, but not on TH-positive nerves. TRPA1 was also located in vimentin-positive interstitial cells. In functional experiments, neither of the TRPA1-agonists (1-100 μM) had any direct effects on ureter tension (baseline/potassium-induced contractions). However, CA, AI, NaHS, and capsaicin (10 μM) decreased (P < 0.01-0.05) tetrodotoxin-sensitive electrically induced (2,4,8,16,32 Hz) contractions. Inhibitory activities were 50-61% (CA), 30-56% (AI), 30-40% (NaHS), and 37-67% (Capsaicin). In the human ureter, TRPA1 is located to sensory nerves and interstitial cells. TRPA1 agonists inhibited electrically induced contractions but had no direct effect on smooth muscle tension of the human ureter. A role for TRPA1 in modulating neurotransmission and possibly peristalsis of the human ureter is proposed.

Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

BackgroundRecent genome-wide association studies have identified transient receptor potential M8 (TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons.MethodsWe investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura.ResultsWe found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker.ConclusionsOur findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.

Electroacupuncture Attenuates CFA-induced Inflammatory Pain by suppressing Nav1.8 through S100B, TRPV1, Opioid, and Adenosine Pathways in Mice

Pain is associated with several conditions, such as inflammation, that result from altered peripheral nerve properties. Electroacupuncture (EA) is a common Chinese clinical medical technology used for pain management. Using an inflammatory pain mouse model, we investigated the effects of EA on the regulation of neurons, microglia, and related molecules. Complete Freund’s adjuvant (CFA) injections produced a significant mechanical and thermal hyperalgesia that was reversed by EA or a transient receptor potential V1 (TRPV1) gene deletion. The expression of the astrocytic marker glial fibrillary acidic protein (GFAP), the microglial marker Iba-1, S100B, receptor for advanced glycation end-products (RAGE), TRPV1, and other related molecules was dramatically increased in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of CFA-treated mice. This effect was reversed by EA and TRPV1 gene deletion. In addition, endomorphin (EM) and N6-cyclopentyladenosine (CPA) administration reliably reduced mechanical and thermal hyperalgesia, thereby suggesting the involvement of opioid and adenosine receptors. Furthermore, blocking of opioid and adenosine A1 receptors reversed the analgesic effects of EA. Our study illustrates the substantial therapeutic effects of EA against inflammatory pain and provides a novel and detailed mechanism underlying EA-mediated analgesia via neuronal and non-neuronal pathways.

The effect of intra-articular vanilloid receptor agonists on pain behavior measures in a murine model of acute monoarthritis.

Arthritis is the most common cause of disability in the US, and the primary manifestation of arthritis is joint pain that leads to progressive physical limitation, disability, morbidity, and increased health care utilization. Capsaicin (CAP) is a vanilloid agonist that causes substance P depletion by interacting with vanilloid receptor transient receptor potential V1 on small unmyelinated C fibers. It has been used topically for analgesia in osteoarthritis with variable success. Resiniferatoxin (RTX) is an ultra potent CAP analog. The aim of this study was to measure the analgesic effects of intra-articular (IA) administration of CAP and RTX in experimental acute inflammatory arthritis in mice. Evoked pain score (EPS) and a dynamic weight bearing (DWB) device were used to measure nociceptive behaviors in a murine model of acute inflammatory monoarthritis. A total of 56 C57B16 male mice underwent EPS and DWB testing – 24 nonarthritic controls and 32 mice with carrageenan-induced arthritis. The effects of pretreatment with 0.1% CAP, 0.0003% RTX, or 0.001% RTX were measured. Nociception was reproducibly demonstrated by increased EPS and reduced DWB measures in the affected limb of arthritic mice. Pretreatment with 0.001% RTX resulted in statistically significant improvement in EPS and DWB measures when compared with those observed in carrageenan-induced arthritis animals. Pretreatment with IA 0.0003% RTX and IA 0.01% CAP resulted in improvement in some but not all of these measures. The remaining 24 mice underwent evaluation following treatment with 0.1% CAP, 0.0003% RTX, or 0.001% RTX, and the results obtained were similar to that of naïve, nonarthritic mice.

Neuro-immune interactions in chemical-induced airway hyperreactivity.

Asthma may be induced by chemical sensitisers, via mechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivo model, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient Kit(Wsh) (/Wsh) mice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

(372) Sex differences in peripheral serotonergic pain processing in male and female rats

Many persistent pain conditions occur predominantly in women making pain a major women’s health issue. As the majority of pain research has been conducted in males, the mechanisms underlying pain in women are unclear. One theory for the prevalence of pain disorders in females is steroid hormone modulation of pain mechanisms. The monoamine neurotransmitter serotonin (5HT) has been highly implicated in various pain conditions that are more prevalent in women. Our recent studies found that peripheral 5HT (1) evokes pain via a subpopulation of trigeminal nociceptors expressing the transient receptor potential V1 channel (TRPV1) and (2) potentiates capsaicin-evoked proinflammatory peptide release from human tooth pulp during the luteal phase of the menstrual cycle. No studies have yet examined the effect of hormone modulation on 5HT-evoked pain in females. We hypothesized that peripheral 5HT evokes greater pain behaviors in female rodents during stages of the estrous cycle when steroid hormones are fluctuating. Female Sprague-Dawley rats (250-350 g) from each stage of the estrous cycle, ovariectomized rats, and intact males were acclimated to the testing apparatuses. Rats received a hindpaw injection of 5HT (2 μg / 100uL; ipl) or saline (n = 6 / group) and thermal hyperalgesia and mechanical allodynia were measured at 10, 20 and 30 minutes. Here we report that female rats in proestrus and estrus exhibited significantly greater and longer-lasting pain behaviors following 5HT injection compared to males, diestrus females, and ovariectomized females, while there was no significant change in edema. These data implicate a modulatory role of hormones on 5HT-evoked pain processing, which may underlie the greater prevalence of pain disorders in women. Our current studies are elucidating the potential enhancing effect of 17-β-estradiol treatment on serotonergic potentiation of the TRPV1 population of sensory neurons in culture. Supported by a Research Enhancement Grant from Texas Woman’s University.

Differing roles for parvalbumin neurons after nerve injury.

Neuropathic pain is abnormal, persistent pain that is caused by injury to the somatosensory system. It commonly involves damage to the peripheral nerves as a result of physical trauma, metabolic diseases such as diabetes, infections such as HIV or shingles, or toxicity induced neuropathies as a result of cancer treatments. Neuropathic pain is common, with epidemiological studies suggesting that around 18–30% of American, European and Australian adults suffer from chronic pain. This leads to significant disabilities and suffering. People with chronic pain complain of spontaneous pains that may be electric-shock like or continuous. Often coexisting with these are abnormal, evoked sensations such as tactile allodynia (pain in response to innocuous stimuli e.g., light brush or pressure) and cold allodynia (pain in response to innocuous cooling), or hyperalgesia (increase pain in response to an already noxious stimulus), as well as ataxia and proprioceptive deficits. The management of these conditions is often suboptimal for two main reasons: firstly, an incomplete knowledge of the mechanisms that are involved (and which of them are the most important). Secondly, patients often have compliance issues with pharmacological medications (Attal, 2012). Both peripheral and central neuronal mechanisms contribute to the pathophysiology and since the turn of the century particular emphasis has focussed on the role of glial cells in pain (West et al., 2015).

Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1) channels

Non-selective transient receptor potential vanilloid (TRPV) cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM) is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1) and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM) and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i), while treatment with THAM (pH 8.5) alone had no effect. However, treatment with THAM (pH 8.5) following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.