Receptor Potential Cation Channel Vanilloid Research Articles

Capsaicin, The Vanilloid Receptor TRPV1 Agonist in Neuroprotection: Mechanisms Involved and Significance.

Hot peppers, also called chilli, chilli pepper, or paprika of the plant genus Capsicum (family Solanaceae), are one of the most used vegetables and spices worldwide. Capsaicin (8-methyl N-vanillyl-6-noneamide) is the main pungent principle of hot green and red peppers. By acting on the capsaicin receptor or transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1), capsaicin selectively stimulates and in high doses defunctionalizes capsaicin-sensitive chemonociceptors with C and Aδ afferent fibers. This channel, which is involved in a wide range of neuronal processes, is expressed in peripheral and central branches of capsaicin-sensitive nociceptive neurons, sensory ganglia, the spinal cord, and different brain regions in neuronal cell bodies, dendrites, astrocytes, and pericytes. Several experimental and clinical studies provided evidence that capsaicin protected against ischaemic or excitotoxic cerebral neuronal injury and may lower the risk of cerebral stroke. By preventing neuronal death, memory impairment and inhibiting the amyloidogenic process, capsaicin may also be beneficial in neurodegenerative disorders such as Parkinson's or Alzheimer's diseases. Capsaicin given in systemic inflammation/sepsis exerted beneficial antioxidant and anti-inflammatory effects while defunctionalization of capsaicin-sensitive vagal afferents has been demonstrated to increase brain oxidative stress. Capsaicin may act in the periphery via the vagal sensory fibers expressing TRPV1 receptors to reduce immune oxidative and inflammatory signalling to the brain. Capsaicin given in small doses has also been reported to inhibit the experimentally-induced epileptic seizures. The aim of this review is to provide a concise account on the most recent findings related to this topic. We attempted to delineate such mechanisms by which capsaicin exerts its neuronal protective effects. We also aimed to provide the reader with the current knowledge on the mechanism of action of capsaicin on sensory receptors.

Mitochondrial Reactive Oxygen Species Mediate Activation of TRPV1 and Calcium Entry Following Peripheral Sensory Axotomy.

Axons that are physically separated from their soma activate a series of signaling events that results in axonal self-destruction. A critical element of this signaling pathway is an intra-axonal calcium rise that occurs just prior to axonal fragmentation. Previous studies have shown that preventing this calcium rise delays the onset of axon fragmentation, yet the ion channels responsible for the influx, and the mechanisms by which they are activated, are largely unknown. Axonal injury can be modeled in vitro by transecting murine dorsal root ganglia (DRG) sensory axons. We coupled transections with intra-axonal calcium imaging and found that Ca2+ influx is sharply reduced in axons lacking trpv1 (for transient receptor potential cation channel vanilloid 1) and in axons treated with capsazepine (CPZ), a TRPV1 antagonist. Sensory neurons from trpv1–/– mice were partially rescued from degeneration after transection, indicating that TRPV1 normally plays a pro-degenerative role after axonal injury. TRPV1 activity can be regulated by direct post-translational modification induced by reactive oxygen species (ROS). Here, we tested the hypothesis that mitochondrial ROS production induced by axotomy is required for TRPV1 activity and subsequent axonal degeneration. We found that reducing mitochondrial depolarization with NAD+ supplementation or scavenging ROS using NAC or MitoQ sharply attenuates TRPV1-dependent calcium influx induced by axotomy. This study shows that ROS-dependent TRPV1 activation is required for Ca2+ entry after axotomy.

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca2+ channel that plays a key role in the active Ca2+ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca2+ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca2+-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca2+ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca2+-selective filter influences the Ca2+ transport function of the TRPV5 channel.Communicated by Ramaswamy H. Sarma

Acupuncture for chronic pain: an update and critical overview.

Acupuncture is now recommended for several chronic pain conditions. Despite supportive evidence of its effectiveness, this ancient approach is often misunderstood, and may still be underused in mainstream practice. A critical review on its effectiveness and practice integration, and mechanisms of action is essential to the medical community that is continuing to seek nonopioid therapies for chronic pain. Mounting evidence supports the effectiveness of acupuncture to treat chronic low back, neck, shoulder, and knee pain, as well as headaches. Additional data are emerging that support the use of acupuncture as an adjunct or alternative to opioids, and in perioperative settings. Findings related to its mechanisms of action include transient receptor potential cation channel vanilloid 1 activation in the periphery, microglial suppression in the cerebral cortex and spinal cord, and regulation of cytokines and other key inflammatory factors in the spinal cord. Incremental integration of acupuncture into pain medicine practices and training programmes continues to grow. Acupuncture is effective, safe, and cost-effective for treating several chronic pain conditions when performed by well-trained healthcare professionals. Further studies on its use as an adjunct or alternative to opioids, and in perioperative settings are needed.

Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels.

There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 μM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 μM) either alone or together with CAPS (10 μM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 μM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required.

A novel actor in mechanotransduction: transient receptor potential cation channel vanilloid (TRPV) 1 in ventilator‐induced lung injury (VILI)

Study objectiveMechanical ventilation can damage the lung by causing endothelial barrier failure, edema formation and lung injury. Yet, the exact mechanisms underlying the mechanotransduction at the microvascular barrier are still unclear. In previous work, we had identified a critical role of the endothelial Ca2+ channel transient receptor potential vanilloid 4 (TRPV4) in endothelial barrier failure subsequent to mechanical overventilation. Here, we assessed the relevance of TRPV1 as novel regulator in VILI in vivo, and probed for its contribution to TRPV4‐induced Ca2+ signalling in vivo, ex vivo and in vitro.MethodsWild type, TRPV4‐ or TRPV1‐deficient mice were ventilated for 2 h with low (7 mL/kg) and high (20 mL/kg) tidal volumes (LVT/HVT) in vivo. Endothelial [Ca2+]i in isolated‐perfused lungs, inflated with 5 or 15 cmH2O, and in human pulmonary microvascular endothelial cells (HPMVECs) was quantified by real‐time imaging with or without pharmacological inhibition of TRPV1 and TRPV4, or after activation of TRPV1 and/or TRPV4 by capsaicin and/or GSK1016790A, respectively.ResultsTRPV1 or TRPV4 deficiency or inhibition by either SB366791 or HC‐067047, respectively, attenuated VILI, and reduced the endothelial [Ca2+]i response to high pressure inflation ex vivo, with exception of SB366791 treatment, which only diminished the sustained [Ca2+]i response, but not the initial Ca2+ influx. In vitro Ca2+ measurements from HPMVECs showed a characteristic increase in endothelial [Ca2+]i that in response to selective TRPV4, yet activation of TRPV1 alone had no effect. Dual activation of both TRPV channels, however, amplified and prolong the TRPV4‐mediated Ca2+ influx into HPMVECs.ConclusionsHere, we demonstrate a critical role of TRPV1 as amplifier of TRPV4‐mediated [Ca2+]i response and barrier failure for VILI in vivo, ex vivo and in vitro, and identify a novel signalling axis that may present putative pharmacological targets for the prevention or treatment of ventilator‐associated lung disease.

PS 08-15 ORAL DOSE OF CAPSAICIN INCREASES BLOOD PRESSURE IN HEALTHY SUBJECTS.

Objective: Capsaicin, the active pungent of spicy foods, is the endogenous ligand of the transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1). Experimental data show that TRPV1 activation leads to vasodilation, water retention and natriuresis - effects that may reduce blood pressure (BP). Recent data found an association between spicy food consumption and improved outcomes, including cardiovascular disease. Capsaicin-induced BP lowering might explain this protective effect. We therefore investigated the BP effects of TRPV1 activation by capsaicin in healthy subjects. Design and Method: In this double blind, randomized, placebo-controlled, cross-over study, participants received either two doses of capsaicin (5.2–6.2 mg, 81.250–97.200 SHU) or matching placebo in random order on two different study days, separated by at least one week. In the 2-hour follow-up after the first dose, BP, heart rate (HR), cardiac output (CO) and systemic vascular resistance index (SVRI) using Omron and NexfinTM and both plasma and urine sodium were repeatedly measured. To challenge osmoregulation, we studied the same parameters for 4 hours after a water loading test (20 mL/kg in 20 minutes) combined with the second dose. Results: We included 12 healthy male subjects (mean age: 23 years). Relative to placebo, 30 min after first capsaicin dose systolic BP significantly increased (after 2 hours: +2.8 ± 1.1 mmHg; p=0.02), but not diastolic BP (Fig 1A). The second capsaicin dose plus water load increased placebo-subtracted diastolic, but not systolic, BP with 4.2 ± 3.6 mmHg after 30 min (p = 0.04). Compared to placebo, capsaicin did affect neither water balance nor natriuresis (Fig 1B,C). Effects on HR, CO and SVRI did not differ from placebo. Conclusions: In healthy subjects, capsaicin increased BP on the short term. We could not identify changes in systemic vascular resistance or water and sodium balance, which were thought to underlie the hypothesized BP effects. Whether long-term capsaicin has similar effects remains to be determined.

Capsaicin, Nociception and Pain.

Capsaicin, the pungent ingredient of the hot chili pepper, is known to act on the transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1). TRPV1 is involved in somatic and visceral peripheral inflammation, in the modulation of nociceptive inputs to spinal cord and brain stem centers, as well as the integration of diverse painful stimuli. In this review, we first describe the chemical and pharmacological properties of capsaicin and its derivatives in relation to their analgesic properties. We then consider the biochemical and functional characteristics of TRPV1, focusing on its distribution and biological effects within the somatosensory and viscerosensory nociceptive systems. Finally, we discuss the use of capsaicin as an agonist of TRPV1 to model acute inflammation in slices and other ex vivo preparations.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves

Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. Invitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. Invivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin invivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both invitro and invivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons

The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. The generation of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with capsaicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1-ANO1 interaction is a significant pain-enhancing mechanism in the peripheral nervous system.

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

Although cannabinoids, such as Δ(9)-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB2R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.

A sensory neuron–expressed IL-31 receptor mediates T helper cell–dependent itch: Involvement of TRPV1 and TRPA1

Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Transient receptor potential cation channel vanilloid (TRPV) 4 in ventilator‐induced lung injury (VILI)

Study objectiveCa2+ entry via TRPV4 has been implicated in stretch‐induced barrier failure in isolated lungs. Here, we assessed the relevance of TRPV4 in VILI in vivo, and probed for the contribution of serum/glucocorticoid‐regulated kinase (SGK) 1, which phosphorylates TRPV4, and of Ca2+‐activated K+ channels (SK, BK) as putative signalling mechanisms upstream and downstream of TRPV4, respectively.MethodsMale, anesthetized mice were mechanically ventilated for 2 h by low (7 ml/kg) and high (20 ml/kg) tidal volumes (LVT/HVT) in wild type and TRPV4‐deficient mice, and following pharmacological inhibition of SGK1, TRPV4, SK or BK channels.ResultsTRPV4 deficiency or inhibition by HC‐067047 attenuated VILI as demonstrated by reduced lung edema, protein leak, MPO activity, inflammatory cytokine levels, and histological features of lung injury. Inhibition of SK or BK channels by apamin or charybdotoxin attenuated lung edema in HVT mice, yet only charybdotoxin had concomitant anti‐inflammatory effects. Lung edema formation in HVT mice was also largely reduced by the SGK1 inhibitor GSK650394.ConclusionsHere, we demonstrate the critical role of TRPV4 for VILI in vivo, and identify potential upstream (SGK1) and downstream (SK, BK) signalling events that may present putative pharmacological targets for the prevention or treatment of ventilator‐associated lung disease.

Expression of nicotinic acetylcholine receptor subunit mRNA in mouse bladder afferent neurons

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT–PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2–4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR – α3∗, α6∗ and α7∗ – and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP–nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP–nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP–nAChRα3 were observed, with eGFP–nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.

A longitudinal study of sensory biomarkers of progression in patients with diabetic peripheral neuropathy using skin biopsies

We aimed to identify biomarkers in skin punch biopsies that could be used to monitor progression of diabetic peripheral neuropathy (DPN), and, in future studies, to assess the efficacy of agents that may reduce progression. Patients with DPN were studied with clinical assessments, skin biopsies, quantitative sensory testing (QST), histamine-induced skin flare, nerve conduction studies and contact heat-evoked potentials (CHEPS). Skin biopsies were performed on two visits with a 6month interval (n=29 patients) to quantify intraepidermal (IENF) and subepidermal (SENF) nerve fibres immunoreactive for: protein gene product 9.5 (PGP9.5), a pan-neuronal marker; transient receptor potential cation channel vanilloid 1 (TRPV1), the heat and capsaicin receptor; and growth associated protein-43 (GAP-43), a marker of regenerating fibres. The IENF were counted along the length of four non-consecutive sections, and results were expressed as fibres per millimetre length of section. SENF were measured by image analysis, and the area of highlighted immunoreactivity was obtained as a percentage (% area) of the field scanned. QST, skin flare and CHEPS were also performed at the two visits. We found that IENF and SENF were significantly reduced for both PGP9.5 and TRPV1 between the first and second skin biopsy over 6months. The annual rate±standard error of the mean of IENF loss was 3.76±1.46 fibres/mm for PGP9.5, and 3.13±0.58 fibres/mm for TRPV1. The other tests did not show significant changes. Strongly positive GAP-43 nerve fibres were found in deep dermis in the patients with diabetes, even in those with an absence of IENF. We conclude that PGP9.5 and TRPV1 IENF and SENF in skin biopsies are useful markers of progression in DPN, whereas GAP-43 SENF could potentially help detect nerve regeneration in severe neuropathy.

Comparison of the analgesic efficacy of oral ABT-116 administration with that of transmucosal buprenorphine administration in dogs

To evaluate the analgesic efficacy of ABT-116, a transient receptor potential cation channel vanilloid subfamily V member 1 antagonist, and compare it with that of buprenorphine by measurement of mechanical and thermal nociceptive thresholds in dogs. Six 7- to 8-month-old dogs (3 males and 3 females). In a crossover study design, all dogs received ABT-116 (30 mg/kg, PO) and buprenorphine (0.03 mg/kg, orotransmucosally), with each treatment separated by 1 week. Physiologic variables were recorded prior to and 1, 6, and 24 hours after drug administration. Thermal (thoracic) and mechanical (dorsolateral aspect of the radius [proximal] and dorsopalmar aspect of the forefoot [distal]) nociceptive thresholds were assessed prior to (baseline) and 15 minutes and 1, 2, 4, 6, 12, 18, and 24 hours after treatment. Buprenorphine administration resulted in higher overall thermal and proximal mechanical nociceptive thresholds, compared with ABT-116. Distal mechanical nociceptive thresholds after treatment were higher than baseline values for both treatments, but the magnitude of change was greater for buprenorphine at 1 hour after administration. Whereas HR and RR sporadically differed from baseline values after ABT-116 administration, rectal temperature increased from a baseline value of 39 ± 0.2°C (mean ± SD) to a peak of 40.6 ± 0.2°C at 6 hours. In dogs without inflammation or nerve injury, PO administration of ABT-116 did not consistently result in an increase in nociceptive thresholds. However, clinically relevant increases in rectal temperature were identified after ABT-116 administration.

The importance of conventional radiography in the mutational analysis of skeletal dysplasias (the TRPV4 mutational family)

The spondylo and spondylometaphyseal dysplasias (SMDs) are characterized by vertebral changes and metaphyseal abnormalities of the tubular bones, which produce a phenotypic spectrum of disorders from the mild autosomal-dominant brachyolmia to SMD Kozlowski to autosomal-dominant metatropic dysplasia. Investigations have recently drawn on the similar radiographic features of those conditions to define a new family of skeletal dysplasias caused by mutations in the transient receptor potential cation channel vanilloid 4 (TRPV4). This review demonstrates the significance of radiography in the discovery of a new bone dysplasia family due to mutations in a single gene.

Characterization of capsaicin induced responses in mice vas deferens: Evidence of CGRP uptake

Calcitonin gene-related peptide (CGRP) is extensively distributed in primary afferent sensory nerves, including those innervating the genitourinary tract. Capsaicin can stimulate the release of CGRP from intracellular stores of these nerves, but this phenomenon has not been investigated in-depth in isolated preparations. The present study sets out to study and characterize the capsaicin as well as CGRP-induced responses in isolated mouse vas deferens. The effects of capsaicin and CGRP family of peptides were studied on electrically-induced twitch responses in the absence or presence of transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) antagonist and CGRP receptor antagonists. Twitch responses were attenuated by capsaicin (1 nM–30 nM) and CGRP family of peptides. The potency order was CGRP > intermedin-long (IMDL) ~ [Cys(Et) 2,7]αCGRP ~ adrenomedullin (AM) > [Cys(ACM) 2,7]αCGRP > amylin (AMY). These responses were disinhibited by the CGRP receptor antagonists and TRPV1 antagonists. The addition of CGRP receptor antagonists caused a transient potentiation of the twitch response and this potentiation was blocked by pretreatment with capsaicin and enhanced by incubation with exogenous CGRP. During the second consecutive cumulative concentration-response curve with capsaicin, the first phase of concentration-response curve disappeared and this was partially restored when the mouse vas deferens was preincubated with CGRP, suggesting the uptake of exogenous CGRP by nerves. Besides showing capsaicin-induced CGRP releases this study shows that exogenous CGRP can be taken up in vas deferens and can be re-released. CGRP uptake will add another dimension in understanding the homeostasis of this neuropeptide.

Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca 2+] i). These compounds would be expected to activate TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by measuring [Ca 2+] i. When an extracellular or intracellular Ca 2+ source was removed, some agonists such as capsaicin could increase [Ca 2+] i. However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named olvanilate) could not elicit a large increase in [Ca 2+] i in the absence of an extracellular or intracellular Ca 2+ source. These results suggest that highly lipophilic compounds cause only a slight Ca 2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the endoplasmic reticulum.