Alexandru Babes

Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.

Sensory acuity and motor dexterity deteriorate when human limbs cool down, but pain perception persists and cold-induced pain can become excruciating. Evolutionary pressure to enforce protective behaviour requires that damage-sensing neurons (nociceptors) continue to function at low temperatures. Here we show that this goal is achieved by endowing superficial endings of slowly conducting nociceptive fibres with the tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 (ref. 2). This channel is essential for sustained excitability of nociceptors when the skin is cooled. We show that cooling excitable membranes progressively enhances the voltage-dependent slow inactivation of tetrodotoxin-sensitive VGSCs. In contrast, the inactivation properties of Nav1.8 are entirely cold-resistant. Moreover, low temperatures decrease the activation threshold of the sodium currents and increase the membrane resistance, augmenting the voltage change caused by any membrane current. Thus, in the cold, Nav1.8 remains available as the sole electrical impulse generator in nociceptors that transmits nociceptive information to the central nervous system. Consistent with this concept is the observation that Nav1.8-null mutant mice show negligible responses to noxious cold and mechanical stimulation at low temperatures. Our data present strong evidence for a specialized role of Nav1.8 in nociceptors as the critical molecule for the perception of cold pain and pain in the cold.

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction

Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold‐sensitive neurones were pre‐selected by screening for an increase in [Ca2+]i on cooling; 49 % of them were also excited by 0.5 μm capsaicin. Action potentials and voltage‐gated currents of cold‐sensitive neurones were clearly distinct from those of cold‐insensitive neurones. All cold‐sensitive neurones expressed an inward current activated by cold and sensitised by (‐)‐menthol, which was absent from cold‐insensitive neurones. This current was carried mainly by Na+ ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage‐gated Ca2+ entry; a minor fraction of Ca2+ entry was voltage‐independent. Application of (‐)‐menthol shifted the threshold temperatures of the cold‐induced depolarisation and the inward current to the same extent, indicating that the cold‐ and menthol‐activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)‐isomer being a less effective agonist than the (‐)‐isomer. Extracellular Ca2+ modulated the cold‐ and menthol‐activated current in a similar way to its action on intact cold receptors: lowered [Ca2+]o sensitised the current, while raised [Ca2+]o antagonised the menthol‐induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca2+ and was mediated by a rise in [Ca2+]i. This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin‐sensitive neurones, capsaicin application caused a profound depression of the cold‐activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold‐activated current towards warmer temperatures. The current was blocked by 50 μm capsazepine and 100 μm SKF 96365. We conclude that the cold‐ and menthol‐activated current is the major mechanism responsible for cold‐induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

Two populations of cold-sensitive neurons in rat dorsal root ganglia and their modulation by nerve growth factor

Cold sensing in mammals is not completely understood, although significant progress has been made recently with the cloning of two cold‐activated ion channels, TRPM8 and TRPA1. We have used rat DRG neurons in primary culture and calcium fluorimetry to identify distinct populations of cold‐sensitive neurons, which may underlie different functions. Menthol sensitivity clearly separated two classes of cold‐responding neurons. One group was menthol‐sensitive (MS), was activated at warmer temperatures and responded faster and with a larger increase in intracellular calcium concentration during cooling; the fraction of MS neurons in culture and their cold sensitivity were both increased in the presence of nerve growth factor. Neurons in the menthol‐insensitive (MI) group required stronger cooling for activation than MS cells and neither their proportion nor their cold sensitivity were significantly altered by nerve growth factor. The two groups of cold‐sensitive neurons also had different pharmacology. A larger fraction of MS cells were capsaicin‐sensitive and coexpression of menthol and capsaicin sensitivity was observed in the absence of NGF. MI neurons were not stimulated by the super‐cooling agent icilin or by the irritant mustard oil. Taken together these findings support a picture in which TRPM8 is the major player in detecting gentle cooling, while TRPA1 does not seem to be involved in cold sensing by MI neurons, at least in the temperature range between 32 and 12 °C.

TRPA1 and Substance P Mediate Colitis in Mice

BACKGROUND & AIMS The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Desensitization of cold- and menthol-sensitive rat dorsal root ganglion neurones by inflammatory mediators

The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.

A high-threshold heat-activated channel in cultured rat dorsal root ganglion neurons resembles TRPV2 and is blocked by gadolinium

Heat‐activated ion channels from the vanilloid‐type TRP group (TRPV1–4) seem to be central for heat‐sensitivity of nociceptive sensory neurons. Displaying a high‐threshold (> 52 °C) for activation, TRPV2 was proposed to act as a sensor for intense noxious heat in mammalian sensory neurons. However, although TRPV2 is expressed in a distinct population of thinly myelinated primary afferents, a widespread expression in a variety of neuronal and non‐neuronal tissues suggests a more diverse physiological role of TRPV2. In its role as a heat‐sensor, TRPV2 has not been thoroughly characterized in terms of biophysical and pharmacological properties. In the present study, we demonstrate that the features of heterologously expressed rat TRPV2 closely resemble those of high‐threshold heat‐evoked currents in medium‐ and large‐sized capsaicin‐insensitive rat dorsal root ganglion (DRG) neurons. Both in TRPV2‐expressing human embryonic kidney (HEK)293t cells and in DRGs, high‐threshold heat‐currents were sensitized by repeated activation and by the TRPV1–3 agonist, 2‐aminoethoxydiphenyl borate (2‐APB). In addition to a previously described block by ruthenium red, we identified the trivalent cations, lanthanum (La3+) and gadolinium (Gd3+) as potent blockers of TRPV2. Thus, we present a new pharmacological tool to distinguish between heat responses of TRPV2 and the closely related capsaicin‐receptor, TRPV1, which is strongly sensitized by trivalent cations. We demonstrate that self‐sensitization of heat‐evoked currents through TRPV2 does not require extracellular calcium and that TRPV2 can be activated in cell‐free membrane patches in the outside‐out configuration. Taken together our results provide new evidence for a role of TRPV2 in mediating high‐threshold heat responses in a subpopulation of mammalian sensory neurons.

Cooling inhibits capsaicin-induced currents in cultured rat dorsal root ganglion neurones.

Whole-cell and single-channel recordings from rat dorsal root ganglion neurones were used to investigate the temperature dependence of currents through the capsaicin receptor (vanilloid receptor 1, VR1). Reducing the temperature from 31 to 14 degrees C inhibited the current induced by 0.5 microM capsaicin by 80%. The Q(10) (temperature coefficient over a 10 degrees C range) of the whole-cell capsaicin-induced current was 2.3 between 10 and 30 degrees C. Single-channel recordings showed that this inhibition by cooling was due to a marked reduction in the open probability (Q(10)=8.2 between 10 and 30 degrees C). This effect can explain the pain relief and reduction in inflammation caused by strong cooling of the skin.

Acute and chronic effects of neurotrophic factors BDNF and GDNF on responses mediated by thermo-sensitive TRP channels in cultured rat dorsal root ganglion neurons

Neurotrophic factors (NTFs), beside regulating neuronal survival in the central and peripheral nervous system, are also involved in the modulation of neuronal function in the adult animal. Both brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) levels are altered in pathological pain states, and exogenous BDNF and GDNF have multiple effects on pain behavior, depending on the animal model (i.e. inflammatory vs. neuropathic). Thermally gated TRP channels TRPM8, TRPA1 and TRPV1 play a significant role in pain signaling and their pattern and level of expression as well as their biophysical properties are altered in chronic pain states. Our aim was to investigate the effect of long-term and acute exposure to BDNF and GDNF on the functional expression of these thermoTRP channels in cultured rat dorsal root ganglion (DRG) neurons. We found that while BDNF treatment primarily increased the fraction of capsaicin-sensitive (TRPV1-expressing) neurons, GDNF exposure led to an increase in the allyl isothiocyanate (AITC)-responding (TRPA1-expressing) population. Moreover, BDNF treatment increased the amplitude of the response to both AITC and capsaicin. Acute treatment with both NTFs leads to a reduction in the magnitude of tachyphylaxis to noxious stimuli (heat and AITC). Overall, our data provides evidence for a role of BDNF and GDNF in regulating the pattern of expression and level of activity of the transducer channels TRPA1 and TRPV1, leading to enhanced neuronal sensitivity to painful stimuli and increased co-expression of thermoTRP channels.