Transient Receptor Potential Vallinoid Research Articles

TRPV1 on the endings of thin fiber muscle afferents is not needed to evoke the exercise pressor reflex

Laura Anselmi, Victor Ruiz-Velasco, Sean D. Stocker, Shannon P. Higgins, Guillaume P. Ducrocq and Marc P. Kaufman. The role played by the transient receptor potential vallinoid-1 (TRPV1) on the endings of thin fiber muscle afferents in evoking the exercise pressor reflex is controversial. To shed light on this controversy, we used TRPV1-/- rats that were made by a CRISPR-Cas9 deletion to introduce a 26-bp frameshift deletion in exon 3. We compared the magnitude of the reflex between TRPV1 +/+ wild-type rats (WT), TRPV1+/- heterozygous rats (HET), and TRPV1-/- knockout rats (KO). The exercise pressor reflex was evoked by stimulating the tibial nerve (40Hz, 0.01ms, 1.5 times motor threshold) in precollicular decerebrated unanesthetized rats whose femoral arteries were either freely perfused or were occluded for 30 sec before the start of contraction. We found that there was no difference between the magnitude of the reflex in the three groups of rats with freely perfused hindlimbs (p=0.61). Specifically, the exercise pressor reflex averaged 16.4 ± 2.2 mmHg in WT rats (n=9), 21.1 ± 5 mmHg in HET rats (n=9), and 17.7 ± 2.3 mmHg in KO rats (n=9). No difference was also observed among the three groups of rats that received acute femoral artery occlusion (p=0.79). Specifically, the peak exercise pressor reflex averaged 15.3 ± 2.2 mmHg in WT rats (n=4), 19.8 ± 4.7 mmHg in HET rats (n=6), and 19 ± 8 mmHg in KO rats (n=3). Stimulation of the tibial nerve after paralysis of the rats with pancuronium (iv) had trivial effects on arterial pressure indicating that the pressor responses to contraction were not caused by electrical stimulation within the axon of the tibial nerve. Intra-carotid arterial injection of the TRPV1 agonist, capsaicin (0.5 mg), evoked a significant pressor response in the WT rats (34.7 ± 9.2 mmHg) and in the HET rats (36.3 ± 7.2 mmHg), but not in the KO rats (3.2 ± 0.4 mmHg). In electrophysiological studies of dorsal root ganglion cells innervating the gastrocnemius muscles, capsaicin evoked inward currents in the WT and HET rats, but not in the KO rats. Moreover, immunofluorescence revealed the presence of TRPV1 in the DRG of WT, but not in the DRG of KO rats. We conclude that TRPV1 is not needed to evoke the exercise pressor reflex either in rats whose femoral arteries were either freely perfused or were occluded. These results are consistent with our previous finding that pharmacological antagonism of TRPV1 had no effect on the exercise pressor reflex. Supported by HL156594 and HL 156513 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electroacupuncture Alleviates Paclitaxel-Induced Peripheral Neuropathic Pain in Rats via Suppressing TLR4 Signaling and TRPV1 Upregulation in Sensory Neurons.

Paclitaxel-induced peripheral neuropathy is a common adverse effect during paclitaxel treatment resulting in sensory abnormalities and neuropathic pain during chemotherapy and in cancer survivors. Conventional therapies are usually ineffective and possess adverse effects. Here, we examined the effects of electroacupuncture (EA) on a rat model of paclitaxel-induced neuropathic pain and related mechanisms. EA robustly and persistently alleviated paclitaxel-induced pain hypersensitivities. Mechanistically, TLR4 (Toll-Like Receptor 4) and downstream signaling MyD88 (Myeloid Differentiation Primary Response 88) and TRPV1 (Transient Receptor Potential Vallinoid 1) were upregulated in dorsal root ganglion (DRGs) of paclitaxel-treated rats, whereas EA reduced their overexpression. Ca2+ imaging further indicated that TRPV1 channel activity was enhanced in DRG neurons of paclitaxel-treated rats whereas EA suppressed the enhanced TRPV1 channel activity. Pharmacological blocking of TRPV1 mimics the analgesic effects of EA on the pain hypersensitivities, whereas capsaicin reversed EA’s effect. Spinal astrocytes and microglia were activated in paclitaxel-treated rats, whereas EA reduced the activation. These results demonstrated that EA alleviates paclitaxel-induced peripheral neuropathic pain via mechanisms possibly involving suppressing TLR4 signaling and TRPV1 upregulation in DRG neurons, which further result in reduced spinal glia activation. Our work supports EA as a potential alternative therapy for paclitaxel-induced neuropathic pain.

SIRT6 as a potential target for treating insulin resistance

AimsWe aimed to explore the role of SIRT6 in Insulin resistance (IR). We are the first to investigate on this crucial relationship in an obese mouse model fed on a high-fat diet (HFD) and an IR model based on the mature 3T3-L1-derived adipocytes. Main methodsWestern blotting (WB) and qPCR analysis were performed to evaluate the SIRT6 protein and mRNA expressions in HFD mice as well as IR cells. Injection of adenovirus encoding SIRT6 gene in HFD mice and transfection of pcDNA3-SIRT6 in IR cells increased the glucose uptake levels and insulin sensitivity. Key findingsThe positive regulatory effects of SIRT6 on transient receptor potential vallinoid 1 (TRPV1) in IR cells were confirmed by a mechanistic investigation at both protein and mRNA levels. Further, the overexpression of SIRT6 was found to activate the TRPV1/Calcitonin gene-related peptide (CGRP) signaling and upregulate the glucose transporter (GLUT) expression at protein and mRNA levels. Additionally, administration of the TRPV1 antagonist, SB-705498 repressed the insulin sensitivity upregulated by SIRT6 overexpression accompanied with the inhibition of CGRP and decrease in GLUT proportions. The results also showed that TRPV1 agonist, Capsaicin boosted the SIRT6-induced glucose uptake, CGRP production, and GLUT4 levels. SignificanceOverall, SIRT6 was concluded to be involved in the TRPV1-CGRP-GLUT4 signaling axis thus leading to increased glucose uptake and decreased IR in HFD mice and 3T3-L1 adipocytes. Therefore, in terms of obesity and diabetes, SIRT6 is a novel candidate for treating IR.

Role of Keratinocytes in Sensitive Skin.

Sensitive skin is a clinical syndrome defined by the occurrence of unpleasant sensations such as burning, stinging, tingling, pricking, or itching in response to various normally innocuous physical, chemical, and thermal stimuli. These particular symptoms have led the consideration of a potential dysfunction of the intra-epidermal nerve fibers (IENF) that are responsible for pain, temperature, and itch perception. This neuronal hypothesis has just been reinforced by recent studies suggesting that sensitive skin could become assimilated to small fiber neuropathy. Meanwhile, the involvement of keratinocytes, the pre-dominant epidermal cell type, has so far mainly been considered because of their role in the epidermal barrier. However, keratinocytes also express diverse sensory receptors present on sensory neurons, such as receptors of the transient receptor potential (TRP) family, including Transient Receptor Potential Vallinoid 1 (TRPV1), one of the main transducers of painful heat which is also involved in itch transduction, and Transient Receptor Potential Vallinoid 4 (TRPV4) which is depicted as a heat sensor. While TRPV1 and TRPV4 are expressed both by sensory neurons and keratinocytes, it has recently been demonstrated that the specific and selective activation of TRPV1 on keratinocytes is sufficient to induce pain. Similarly, the targeted activation of keratinocyte-expressed TRPV4 elicits itch and the resulting scratching behavior. So, contrary to classical conception, the IENF are not the exclusive transducers of pain and itch. In light of these recent advances, this review proposes to consider the putative role of epidermal keratinocytes in the generation of the unpleasant sensations characteristic of sensitive skin syndrome.

A SNAP-25 cleaving chimera of botulinum neurotoxin /A and /E prevents TNFα−induced elevation of the activities of native TRP channels on early postnatal rat dorsal root ganglion neurons

Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we firstly demonstrate by immuno-cytochemical labelling that TNFα augments the surface content of these channels on rat cultured dorsal root ganglion (DRG) neurons which, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNFα-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) and reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNFα-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNFα-dependent augmented, but not resting, surface trafficking of TRPV1/A1. As TNFα is known to induce nociceptive hyper-sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.

Viscerosensory input drives angiotensin II type 1A receptor-expressing neurons in the solitary tract nucleus.

Homeostatic regulation of visceral organ function requires integrated processing of neural and neurohormonal sensory signals. The nucleus of the solitary tract (NTS) is the primary sensory nucleus for cranial visceral sensory afferents. Angiotensin II (ANG II) is known to modulate peripheral visceral reflexes, in part, by activating ANG II type 1A receptors (AT1AR) in the NTS. AT1AR-expressing NTS neurons occur throughout the NTS with a defined subnuclear distribution, and most of these neurons are depolarized by ANG II. In this study we determined whether AT1AR-expressing NTS neurons receive direct visceral sensory input, and whether this input is modulated by ANG II. Using AT1AR-GFP mice to make targeted whole cell recordings from AT1AR-expressing NTS neurons, we demonstrate that two-thirds (37 of 56) of AT1AR-expressing neurons receive direct excitatory, visceral sensory input. In half of the neurons tested (4 of 8) the excitatory visceral sensory input was significantly reduced by application of the transient receptor potential vallinoid type 1 receptor agonist, capsaicin, indicating AT1AR-expressing neurons can receive either C- or A-fiber-mediated input. Application of ANG II to a subset of second-order AT1AR-expressing neurons did not affect spontaneous, evoked, or asynchronous glutamate release from visceral sensory afferents. Thus it is unlikely that AT1AR-expressing viscerosensory neurons terminate on AT1AR-expressing NTS neurons. Our data suggest that ANG II is likely to modulate multiple visceral sensory modalities by altering the excitability of second-order AT1AR-expressing NTS neurons.

Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells.

The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium–dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.

Fluid-induced lung injury-role of TRPV4 channels.

Administration of bolus intravenous fluid is associated with respiratory dysfunction and increased mortality, findings with no clear mechanistic explanation. The objective of this study was to examine whether bolus intravenous (i.v.) fluid administration results in acute lung injury in a rat model and further, to examine whether this injury is associated with transient receptor potential vallinoid (TRPV)4 channel function and endothelial inflammatory response. Healthy male Sprague-Dawley rats were administered 60ml/kg 0.9% saline i.v. over 30min. Manifestation of acute lung injury was assessed by lung physiology, morphology, and markers of inflammation. The role of TRPV4 channels in fluid-induced lung injury was subsequently examined by the administration of ruthenium red (RR) in this established rat model and again in TRPV4 KO mice. In endothelial cell culture, permeability and P-selectin expression were measured following TRPV4 agonist with and without antagonist; 0.9% saline resulted in an increase in lung water, lavage protein and phospholipase A2, and plasma angiopoietin-2, with worsening in arterial blood oxygen (PaO2), lung elastance, surfactant activity, and lung histological injury score. These effects were ameliorated following i.v. fluid in rats receiving RR. TRPV4 KO mice did not develop lung edema. Expression of P-selectin increased in endothelial cells following administration of a TRPV4 agonist, which was ameliorated by simultaneous addition of RR. Bolus i.v. 0.9% saline resulted in permeability pulmonary edema. Data from ruthenium red, TRPV4 KO mice, and endothelial cell culture suggest activation of TRPV4 and release of angiopoietin 2 and P-selectin as the central mechanism.

Interactions of the Mechanosensitive Channels with Extracellular Matrix, Integrins, and Cytoskeletal Network in Osmosensation.

Life is maintained in a sea water-like internal environment. The homeostasis of this environment is dependent on osmosensory system translation of hydromineral information into osmotic regulatory machinery at system, tissue and cell levels. In the osmosensation, hydromineral information can be converted into cellular reactions through osmoreceptors, which changes thirst and drinking, secretion of antidiuretic vasopressin (VP), reabsorption of water and salt in the kidneys at systemic level as well as cellular metabolic activity and survival status at tissue level. The key feature of osmosensation is the activation of mechanoreceptors or mechanosensors, particularly transient receptor potential vallinoid (TRPV) and canonical (TRPC) family channels, which increases cytosolic Ca2+ levels, activates osmosensory cells including VP neurons and triggers a series of secondary reactions. TRPV channels are sensitive to both hyperosmotic and hyposmotic stimuli while TRPC channels are more sensitive to hyposmotic challenge in neurons. The activation of TRP channels relies on changes in cell volume, membrane stretch and cytoskeletal reorganization as well as hydration status of extracellular matrix (ECM) and activity of integrins. Different families of TRP channels could be activated differently in response to hyperosmotic and hyposmotic stimuli in different spatiotemporal orders, leading to differential reactions of osmosensory cells. Together, they constitute the osmosensory machinery. The activation of this osmoreceptor complex is also associated with the activity of other osmolarity-regulating organelles, such as water channel protein aquaporins, Na-K-2Cl cotransporters, volume-sensitive anion channels, sodium pump and purinergic receptors in addition to intercellular interactions, typically astrocytic neuronal interactions. In this article, we review our current understandings of the composition of osmoreceptors and the processes of osmosensation.

(287) Anoctamin-1 Cl- channel in nociception: inhibition by T16[inh]-A01 and activation by an N-aroylaminothiazole and capsaicin

Anoctamin 1 (ANO1 or TMEM16A) Ca2+−gated Cl− channels of nociceptor sensory neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl− concentrations ([Cl−]i) sensory neuronal Cl− channels, such as ANO1, are excitatory. ANO1 channels are directly activated by an N-aroylaminothiazole (E-act). At physiological [Cl−]i (40mM), E-act (10μM) direct activation of ANO1 increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded using whole-cell electrophysiology of mouse dorsal root ganglia (DRG) sensory neurons. Nocifensive responses of mice hind paws were also dramatically induced by subcutaneous injections of E-act (5mM), which was attenuated by co-injection of ANO1-inhibitor T16A[inh]-A01 (1.3mM). ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. At physiological [Cl−]i, capsaicin (15μM)-induced action potential firing was diminished by co-application with ANO1-inhibitor T16A[inh]-A01 (20μM) To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons. (To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin−B) current-clamp technique). Under conditions of low [Cl−]i (10mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-actcapsaicin (50μM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3mM). In summary, an ANO1-activator induced [Cl−]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. The results indicate that sensory neuronal ANO1 facilitates TRPV1 triggering of action potentials, resulting in enhanced nociception and inhibition of ANO1 could be a novel means of inducing analgesia.

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons

The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system.

Anoctamin-1 Cl(-) channels in nociception: activation by an N-aroylaminothiazole and capsaicin and inhibition by T16A[inh]-A01.

BackgroundAnoctamin 1 (ANO1 or TMEM16A) Ca2+-gated Cl− channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl− concentrations ([Cl−]i) sensory neuronal Cl− channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl−]i and (2) by lowering sensory neuronal [Cl−]i to switch ANO1 to be inhibitory.ResultsANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl−]i (40 mM), E-act (10 µM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin–B) current-clamp technique. Subsequently, at physiological [Cl−]i, capsaicin (15 µM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 µM). Under conditions of low [Cl−]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 µM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM).ConclusionsAn ANO1-activator (E-act) induced [Cl−]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0061-y) contains supplementary material, which is available to authorized users.

Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture.

Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1), a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture.

The Anti-Inflammatory Effects of Acupuncture and Their Relevance to Allergic Rhinitis: A Narrative Review and Proposed Model

Classical literature indicates that acupuncture has been used for millennia to treat numerous inflammatory conditions, including allergic rhinitis. Recent research has examined some of the mechanisms underpinning acupuncture's anti-inflammatory effects which include mediation by sympathetic and parasympathetic pathways. The hypothalamus-pituitary-adrenal (HPA) axis has been reported to mediate the antioedema effects of acupuncture, but not antihyperalgesic actions during inflammation. Other reported anti-inflammatory effects of acupuncture include an antihistamine action and downregulation of proinflammatory cytokines (such as TNF-α, IL-1β, IL-6, and IL-10), proinflammatory neuropeptides (such as SP, CGRP, and VIP), and neurotrophins (such as NGF and BDNF) which can enhance and prolong inflammatory response. Acupuncture has been reported to suppress the expression of COX-1, COX-2, and iNOS during experimentally induced inflammation. Downregulation of the expression and sensitivity of the transient receptor potential vallinoid 1 (TRPV1) after acupuncture has been reported. In summary, acupuncture may exert anti-inflammatory effects through a complex neuro-endocrino-immunological network of actions. Many of these generic anti-inflammatory effects of acupuncture are of direct relevance to allergic rhinitis; however, more research is needed to elucidate specifically how immune mechanisms might be modulated by acupuncture in allergic rhinitis, and to this end a proposed model is offered to guide further research.

Isolation of single cells from murine late distal convoluted tubules and connecting tubules

Proteomic studies of late distal convoluted tubule (DCT2) and connecting tubule (CNT) cells have so far been hindered by technical difficulties in isolating these cells with sufficient yield and purity. We exploited a mouse model with endogenous expression of enhanced green fluorescent protein (EGFP) driven by the transient receptor potential vallinoid 5 (TRPV5) channel promoter. Whole kidneys were minced and incubated with 1 mg/ml proteinase K and 1 mg/ml collagenase II for 45 min at 37°C agitated at 180 RPM. The tubule‐suspension was treated with 0.12% trypsin in Ca2+‐free buffer for 2 × 5 min to obtain single cells. Cell separation and morphology were evaluated by light microscopy. Blood vessels and cell clusters were removed by passing the cell suspension through a 40‐μm mesh before fluorescence activated cell sorting. Cells were sorted on a FACS AriaIII (BD) with 100 μm nozzle at 20 psi and 488 nm excitation. Dead cells (approximately 2%) were excluded using propidium iodide. By this method, the yield corresponded to approximately 2.5×107 cells per mouse kidney of which 6×105 were GFP positive (2.4% of all cells) or 38 μg protein per kidney. Thus, we have developed a protocol that may allow proteomic analysis of mouse DCT2/CNT cells with virtually no contamination with cells from other renal structures or with intercalated cells from DCT2/CNT. Supported by the Danish Council for Independent Research | Medical Sciences.

Reduced penetrance in hereditary motor neuropathy caused by TRPV4 Arg269Cys mutation

Incomplete penetrance has rarely been reported in Charcot-Marie-Tooth disease. Our aim is to describe reduced penetrance in a hereditary motor neuropathy pedigree due to mutation in the transient receptor potential vallinoid 4 (TRPV4) gene. The pedigree comprised two affected members, the proband aged 44 years and her affected daughter aged 7 years, and seven additional related subjects, three of whom were subclinical gene mutation carriers aged 9, 40 and 70 years. Clinico-electrophysiological studies, MRI of lower-limb musculature and genetic testing of the TRPV4 were performed. The proband presented with a moderate facio-scapulo-peroneal syndrome, whereas her symptomatic daughter suffered from severe congenital spinal muscular atrophy with arthrogryposis, laryngomalacia, and vocal cord paresis. Electrophysiological evaluation revealed a pure motor axonal neuropathy. In the proband, MRI showed extensive and widespread fatty atrophy of lower-leg musculature, whereas in thigh musculature there was just mild distal fatty infiltration of vastus lateralis. Genetic testing revealed a heterozygous Arg269Cys mutation in the TPRV4 gene. In all three mutation carriers results from clinical and electrophysiological examination, and MRI of foot and lower-leg musculature were normal. We conclude that non-penetrance may be an integral feature of neuropathic syndromes associated with TRPV4 gene mutation.

Acute capsaicin treatment reduces myocardial infarct size in rats via the transient receptor potential vallinoid 1 channel

Acute capsaicin treatment reduces myocardial infarct size in rats via the transient receptor potential vallinoid 1 channel

TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress

The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca(2+) entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca(2+) and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not TRPV4(-/-) mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4(-/-) mice compared with WT controls. Using a fura-2/Mn(2+) quenching assay, shear was observed to produce rapid Ca(2+) influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated Ca(2+) entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into Ca(2+) signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries.

Role of TRPV4 channels in agonist‐induced endothelial Ca2+ entry and vasodilation: Evidence from TRPV4‐deficient mice

Transient receptor potential vallinoid 4 (TRPV‐4), is a Ca2+‐permeant cation channel implicated in endothelial‐dependent vasorelaxation to mechanical stimuli but limited information exists for agonist‐induced responses. We hypothesized that TRPV‐4 is required for agonist‐induced vasodilation by regulating Ca2+ entry. Isometric tension was measured in isolated mesenteric arteries (MA) from wild type (WT) and TRPV‐4 knockout mice using a wire myograph. Dilation to acetylcholine (Ach, 10−9–10−5 M) was diminished in TRPV‐4−/− vs. WT MA (% max relaxation, 44±12 and 86±5, respectively n=4, P<0.05). L‐NAME, blocked Ach relaxation in TRPV‐4−/− and WT MA (11±4 and 35±11, respectively n=4, P<0.05). Responses to papaverine were similar in WT and TRPV‐4−/− MA. Intracellular calcium ([Ca2+]i) was assessed after intraluminal fura‐2 (fluorescence) in MA. Ach (10−5 M) increased endothelial [Ca2+]i in WT and TRPV‐4−/− MA (Δ[Ca2+]i, 49±4.6nM and 26±2.1nM, respectively n=4). The addition of 4α‐PDD, a TRPV4 opener, increased [Ca2+]i in WT but not in TRPV‐4−/− (Δ[Ca2+]i, 164±30nM and 15±1.3nM, respectively n=4). In endothelium of WT MA the increase in [Ca2+] was abolished by the TRPV‐4 blocker ruthenium red (Δ[Ca2+]i, RR, 19±1.2nM n=4). These data suggest an important role for the TRPV‐4 channels in vasodilation to Ach, and regulation of endothelial intracellular Ca2+in mice MA.