Menthacarin induces calcium ion influx in sensory neurons, macrophages and colonic organoids of mice.

AIMS: Menthacarin is a herbal combination that is clinically used for the treatment of functional gastrointestinal disorders (FGIDs). In several clinical studies, Menthacarin reduced visceral hypersensitivity-related symptoms. Pathogenesis of visceral hypersensitivity is multifactorial. This involves several cell types and different transient receptor potential ion channels (TRPs); these ion channels are highly conductive for calcium ions. Since transient changes in cytosolic calcium levels are crucial for many functions of living cells, we investigated if Menthacarin can induce calcium influx in sensory, largely nociceptive, neurons from dorsal root ganglia (DRG), peritoneal macrophages (PMs) and colonic organoids. MAIN METHODS: We employed the calcium imaging technique on sensory neurons from DRG, PMs and colonic organoids isolated from mice. All cells were superfused by Menthacarin at several concentrations (600, 1200, 1800 µg/ml) during the experiments, followed by calcium ionophor ionomycin (Iono., 1 µM) as a positive control. KEY FINDINGS: Menthacarin induced concentration-dependent calcium ion influx in all investigated cell types. Furthermore, repeated applications of Menthacarin induced tachyphylaxis (desensitisation) of calcium responses in sensory neurons and colonic organoids. SIGNIFICANCE: Menthacarin-induced calcium influx into sensory neurons, macrophages and colonic organoids is probably related to its clinical desensitising effects in patients with FGIDs.

Dual opposing actions of STW 5 on TRP receptors mediate neuronal desensitisation in vitro.

AIMS: STW 5 is an herbal drug combination used for the treatment of functional gastrointestinal disorders (FGIDs) with visceral hypersensitivity as the therapy-resistant hallmark. STW 5 has been clinically proven to alleviate visceral hypersensitivity-related symptoms, including abdominal pain, bloating, nausea, and early satiety. However, the molecular and cellular mechanisms underlying the antinociceptive action of STW 5 remain unknown. Here, we investigate the role of STW 5 in the calcium mobilisation of dorsal root ganglion (DRG) sensory neurons. MAIN METHODS: Calcium imaging experiments were performed with freshly dissociated cultured murine DRG neurons isolated from mice by microfluorometry. TRPA1-deficient DRGs, TRPV1-deficient DRGs, TRPA1/V1 double-deficient DRGs, and wild-type DRGs have been used to investigate the role of TRPs ion channels in mediating STW 5 action. KEY FINDINGS: STW 5 (1.74 and 5.8 mg/ml) induced calcium ion influx into DRG neurons in a concentration-dependent manner. Calcium transients were desensitised during repeated exposure to STW 5, an effect that was facilitated in TRPA1-deficient DRGs and less pronounced in TRPV1-deficient DRGs compared to wild-type (WT) DRGs. SIGNIFICANCE: Repeated exposure to STW 5 induced desensitisation of sensory neurons and may ultimately contribute to its proven clinical efficacy against sensory-related symptoms in patients with FGID, including abdominal pain, bloating, nausea, and early satiety. This effect is modulated by the two prominent irritant sensors in nociceptors, TRPA1 and TRPV1.

Transient receptor potential melastatin 8 ion channel in macrophages modulates colitis through a balance-shift in TNF-alpha and interleukin-10 production.

The transient receptor potential (TRP) ion channel family is well characterized in sensory neurons; however, little is known about its role in the immune system. Here we show that the cold-sensing TRPM8 has an unexpected role in innate immunity. TRPM8 expression and function in macrophages were demonstrated in vitro using molecular techniques and calcium imaging. In addition, adoptive macrophage transfer and systemic interleukin (IL)-10 overexpression were performed in experimental colitis. TRPM8 activation induced calcium-transients in murine peritoneal macrophages (PM) and bone marrow-derived macrophages of wild-type (WT) but not TRPM8-deficient mice. TRPM8-deficient PM exhibited defective phagocytosis and increased motility compared with those in WT, whereas the opposite effects of TRPM8 activation were induced in WT PM. TRPM8 activation or blockage/genetic deletion induced a anti- or pro-inflammatory macrophage cytokine profile, respectively. WT mice treated with repeated menthol (TRPM8 agonist) enemas were consistently protected from experimental colitis, whereas TRPM8-deficient mice showed increased colitis susceptibility. Adoptive transfer of TRPM8-deficient macrophages aggravated colitis, whereas systemic IL-10 overexpression rescued this phenotype. TRPM8 activation in peptidergic sensory neurons did not affect neuropeptide release from the inflamed colon. TRPM8 in macrophages determines pro- or anti-inflammatory actions by regulating tumor necrosis factor-α and interleukin-10 production. These findings suggest novel TRPM8-based options for immunomodulatory intervention.

The prokineticin Bv8 sensitizes cutaneous terminals of female mice to heat.

BACKGROUND: Injection of the noxious peptide Bv8 has previously been shown to induce a biphasic thermal hyperalgesia in rodents, the first peak presumably due to peripheral sensitization. This hypothesis has never been directly confirmed. We have assessed whether Bv8 can indeed sensitize peripheral nerve fibres in the mouse to heat. METHODS: We used recordings from single cutaneous fibres, cutaneous calcitonin gene-related peptide (CGRP) release and immunostaining in nerves and plantar skin to evaluate the Bv8 effects on cutaneous nerves. RESULTS: Application of Bv8 at nanomolar concentrations (30-310 nmol/L) to skin preparations significantly increased the heat-induced discharge, the heat-induced afterdischarge and reduced threshold temperature of single unmyelinated polymodal fibres. Furthermore, application of Bv8 to hind-paw skin flaps or trigeminal ganglia significantly elevated their heat-induced CGRP release. Capsaicin-induced and to a lesser extent also KCl-induced CGRP releases were also augmented after Bv8 treatment. Immunohistochemistry revealed co-localization of prokineticin 2 (Bv8 ortholog in rodents) and CGRP in both plantar skin and nerve tissues. These results confirm that Bv8 sensitizes cutaneous nerve endings to heat, partly, although not exclusively through TRPV1 activation. CONCLUSION: Our results thus support the hypothesis that the first hyperalgesic phase to follow Bv8 injection to hind paws of intact animals is due to peripheral sensitization of nociceptors. WHAT DOES THIS STUDY ADD?: Our data provide mechanistic insights into the effect Bv8 application exerts on afferent nerve endings and into the concomitant development of thermal hyperalgesia.

Mice and rats differ with respect to activity-dependent slowing of conduction velocity in the saphenous peripheral nerve.

We assess in mice, the electrophysiological criteria developed in humans and rats in vivo for unmyelinated (C) fibre differentiation into sub-classes, derived from the activity-induced latency increase ("slowing") in response to electrical stimulation during 6 min at 0.25 Hz followed by 3 min at 2 Hz. Fibres are considered nociceptors if they show more than 10% slowing at 2 Hz; nociceptors are further divided into mechanosensitive ("polymodal") and mechanoinsensitive ("silent") ones according to a latency shift of less and more than 1% during the first minute at 0.25 Hz, respectively. Sympathetic postganglionics are recognised by 2-10% slowing at 2 Hz; units slowing less than 2% at 2 Hz remain uncategorised. For assessment of these criteria, we also developed a novel in vivo technique for recording of peripheral single-fibres in the mouse. We compared the theoretical slowing-rate discriminator criteria with experimental data obtained from mice in vivo/in vitro and rats in vitro. Out of 69 cutaneous mouse C-fibres in vitro and 19 in vivo, only 38 (67%) and 9 (47%) met the above 1% criterion, respectively; sympathetics were not identified. In contrast, out of 20 rats nerve fibres in vitro, 19 (95%) met this criterion. We conclude that (A) our novel electrophysiological technique is a practical method for examining mouse cutaneous single-fibres in vivo and (B) the published criterion for identifying silent nociceptors in rats and humans is not applicable in mice.

A new paradigm to understand and treat diabetic neuropathy.

The clinical symptoms of diabetic neuropathy (DN) manifest in a time dependent manner as a positive symptoms (i. e. pain, hypersensitivity, tingling, cramps, cold feet etc.) during its early stages and by a loss of function (i. e. loss of sensory perception, delayed wound healing etc.) predominating in the later stages. Elevated blood glucose alone cannot explain the development and progression of DN and the lowering of blood glucose is insufficient in preventing and/or reversing neuropathy in patients with type 2 diabetes. Recently it has been shown that the endogenous reactive metabolite methylglyoxal (MG) can contribute to the gain of function via post-translational modification in DN of neuronal ion channels involved in chemosensing and action potential generation in nociceptive nerve endings. Dicarbonyls, such as MG, that are elevated in diabetic patients, modify DNA as well as extra- and intracellular proteins, leading to the formation of advanced glycation endproducts (AGEs). Increased formation of AGEs leads to increased cellular stress, dysfunction and ultimately cell death. The interaction of AGE-modified proteins through cell surface receptors, such as RAGE, can lead to increased cellular activation and sustained inflammatory responses, which are the molecular hallmarks of the later, degenerative, stages of DN. The direct and indirect effects of dicarbonyls on nerves or neuronal microvascular network provides a unifying mechanism for the development and progression of DN. Targeting the accumulation of MG and/or prevention of RAGE interactions may therefore provide new, more effective, therapeutic approaches for the treatment of DN.

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block.

BACKGROUND AND PURPOSE: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. EXPERIMENTAL APPROACH: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. KEY RESULTS: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. CONCLUSIONS AND IMPLICATIONS: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.

TRPA1 and TRPV1 are differentially involved in heat nociception of mice.

BACKGROUND: Two transient receptor potential (TRP) channels, TRPV1 and TRPA1, have been physiologically studied with regard to noxious heat transduction. Evidence argues against these channels as sole transducers of noxious heat or cold, respectively. Moreover, in submammalian species the TRPA1 orthologue shows heat sensitivity. METHODS: In vitro, single-fibre and compound action potential recordings from C-fibres as well as measurements of stimulated cutaneous CGRP release are combined with behavioural experiments to assess heat responsiveness in wild type mice, TRPA1 and TRPV1 as well as double-null mutants. RESULTS: Heat thresholds of cutaneous C-mechano-heat sensitive fibres were significantly higher in TRPA1-/- (43 °C) than +/+ (40 °C) mice, and averaged heat responses were clearly weaker, whereas TRPV1-/- showed normal heat thresholds and responses (up to 46 °C). Compound action potential recordings revealed much less activity-dependent slowing of conduction velocity upon noxious heat stimulation in TRPA1-/- and a delayed deficit in TRPV1-/- in comparison to controls. Heat-induced calcitonin gene-related peptide release was reduced in TRPV1-/- but not TRPA1-/- animals. Paw withdrawal latencies to radiant heat were significantly elevated in TRPA1-/-, more so in TRPV1-/- animals. In general, double-null mutants were similar to TRPV1-/- except for the single-fibre heat responses which appeared as weak as in TRPA1-/-. CONCLUSIONS: Our results indicate that in addition to TRPV1, TRPA1 plays a role in heat nociception, in particular in definition of the heat threshold, and might therefore serve as a therapeutic target in acute inflammatory pain.

TRPV1, TRPA1, and CB1 in the isolated vagus nerve--axonal chemosensitivity and control of neuropeptide release.

Vagal sensory afferents innervating airways and abdominal tissues express TRPV1 and TRPA1, two depolarizing calcium permeable ion channels playing a major role in sensing environmental irritants and endogenous metabolites which cause neuropeptide release and neurogenic inflammation. Here we have studied axonal chemosensitivity and control of neuropeptide release from the isolated rat and mouse vagus nerve by using prototypical agonists of these transduction channels - capsaicin, mustard oil and the specific endogenous activators, anandamide (methyl arachidonyl ethanolamide, mAEA), and acrolein, respectively. Capsaicin evoked iCGRP release from the rat vagus nerve with an EC50 of 0.12 µM. Co-application of mAEA had a dual effect: nanomolar concentrations of mAEA (0.01 µM) significantly reduced capsaicin-evoked iCGRP release while concentrations ≥ 1 µM mAEA had sensitizing effects. Only 100 µM mAEA directly augmented iCGRP release by itself. In the mouse, 310 µM mAEA increased release in wildtype and TRPA1-/- mice which could be inhibited by capsazepine (10 µM) and was completely absent in TRPV1-/- mice. CB1-/- and CB1/CB2 double -/- mice equally displayed increased sensitivity to mAEA (100 µM) and a sensitizing effect to capsaicin, in contrast to wildtypes. Acrolein and mustard oil (MO)--at µM concentrations--induced a TRPA1-dependent iCGRP release; however, millimolar concentrations of mustard oil (>1mM) evoked iCGRP release by activating TRPV1, confirming recent evidence for TRPV1 agonism of high mustard oil concentrations. Taken together, we present evidence for functional expression of excitatory TRPV1, TRPA1, and inhibitory CB1 receptors along the sensory fibers of the vagus nerve which lend pathophysiological relevance to the axonal membrane and the control of neuropeptide release that may become important in cases of inflammation or neuropathy. Sensitization and possible ectopic discharge may contribute to the development of autonomic dysregulation in visceral tissues that are innervated by the vagus nerve.

Sensitized peripheral nociception in experimental diabetes of the rat.

Painful neuropathy is a common complication of diabetes. Particularly in the early stage of diabetic neuropathy, patients are characterized by burning feet, hyperalgesia to heat, and mechanical stimuli, as if residual nociceptors were sensitized. Such symptoms are barely explained by common pathophysiological concepts of diabetic neuropathy. Diabetes was induced in Wistar rats by streptozotocin (STZ). After 4 weeks behavioral testing (Plantar test, Randall-Selitto) was conducted. Basal and stimulated release of calcitonin gene-related peptide (CGRP), Substance P (SP) and prostaglandin E(2) (PGE(2)) from isolated skin and sciatic nerve were assessed by enzyme immunoassays. Electrophysiological properties of identified nociceptors under hyperglycemic, hypoxic, and acidotic conditions were investigated using the skin-nerve preparation. The diabetic rats showed hyperalgesia to heat and pressure stimulation. The basal CGRP/SP release was reduced, but chemical stimulation with bradykinin induced greater release of SP, CGRP and PGE(2) than in control animals. In contrast, capsaicin-stimulated CGRP release was reduced in sciatic nerves. Hypoxia per se lowered von Frey thresholds of most C-nociceptors to half. Hyperglycemic hypoxia induced ongoing discharge in all diabetic but not control C-fibers which was further enhanced under acidosis. Sensory and neurosecretory nociceptor functions are sensitized in diabetes. Diabetic C-fibers show exaggerated sensitivity to hyperglycemic hypoxia with and without additional acidosis, conditions that are thought to mimic ischemic episodes in diabetic nerves. Ongoing C-fiber discharge is known to induce spinal sensitization. Together with altered receptor and ion channel expressions this may contribute to painful episodes in diabetic neuropathy.

Acid-induced CGRP release from the stomach does not depend on TRPV1 or ASIC3.

BACKGROUND: Acid-sensing and regulating reactions are vitally important in the upper gastrointestinal tract and disturbances are common. Sensory neurons in the mucosa detect the intrusion of hydrogen ions and, by their release of vasoactive neuropeptides, seem to play a predominantly protective role in these tissues. METHODS: The model to investigate sensory transduction of proton stimuli in the isolated everted mouse stomach was to measure the induced calcitonin gene-related peptide (CGRP) release as an index of neuronal activation. KEY RESULTS: Proton concentrations in the range of pH 2.5-0.5 stimulated the release of CGRP and substance P and profoundly decreased the prostaglandin E2 formation in outbred CD mice. A similar linearly pH-dependent CGRP release was observed in inbred C57BL/6 mice, fully dependent on extracellular calcium at pH 2, partially at pH 1. Both transient receptor potential vanilloid type 1 (TRPV1) and acid-sensing ion channel type 3 (ASIC3) are expressed in the sensory neurons innervating the stomach walls and are responsible for the transduction of acidic stimuli in other visceral organs. However, the proton-induced gastric CGRP release in mice lacking the TRPV1 or the ASIC3 receptor-channels was the same as in corresponding wild-type mice. Nonetheless, the pharmacological blockers N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide and amiloride, respectively, inhibited the acid-stimulated CGRP release, although to the same extend in wild types as TRPV1 and ASIC3 knockout mice. CONCLUSIONS & INFERENCES: Adequate proton concentrations inhibit prostaglandin and stimulate CGRP release from the stomach wall, however, the transduction mechanism in the gastric sensory neurons remains unclear.

Projected pain from noxious heat stimulation of an exposed peripheral nerve--a case report.

Distinct sensory properties of unmyelinated axons in the isolated rat sciatic nerve have previously been revealed by measuring stimulated neuropeptide (CGRP) release in response to noxious stimuli. Axonal sensitization to heat by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel TRPV1. Recently, we have demonstrated in vitro that heat stimulation of nociceptive axons generates ectopic action potential discharge which resembles the heat response of the corresponding cutaneous nerve endings. It remained however, to be established whether adequate axonal stimulation could also generate projected sensations in a conscious human subject. In a singular human trial, the superficial radial nerve (SR) was exposed and stimulated mechanically as well as with noxious cold (3 degrees C). These stimuli were unable to induce any conscious local or projected sensations. However, controlled radiant heat applied to the nerve resulted in intense slowly adapting burning pain sensations projected into the center of the SR innervation area. No local sensation was reported. Thus, presumably activated nervi nevorum in the sheath of a healthy nerve do not cause conscious sensations, while axons of passage in mid-nerve exhibit a sensory transduction capacity for noxious heat though not for mechanical and cold stimulation. Axonal heat transduction may therefore become a source of ectopic discharge and neuropathic pain when heat threshold drops to body temperature as is the case with peripheral nerve endings in inflamed skin.

Mechanisms of potassium- and capsaicin-induced axonal calcitonin gene-related peptide release: involvement of L- and T-type calcium channels and TRPV1 but not sodium channels.

We have previously shown that capsaicin, noxious heat, protons and potassium ions (K(+)) induce a graded, calcium- and receptor-dependent increase of immunoreactive calcitonin gene-related peptide (iCGRP) release from isolated rat sciatic axons. Morphological evidence for axonal vesicular exocytosis has also been presented. Here we determine the differential contribution of voltage-gated calcium and sodium channels to high extracellular potassium and capsaicin-induced iCGRP secretion. Blockade of L-type calcium channels significantly decreased the K(+)-induced axonal response (nimodipine (10 microM) by 66% and methoxyverapamil, D600 (50 microM), by 77%). Interestingly, however, D600 was unable to reduce the capsaicin-induced iCGRP release. Omega-Conotoxin GVIA (1 microM), a N-type blocker, and omega-agatoxin TK (0.1 microM), a P/Q-type blocker, had no significant effect. Also the anticonvulsant gabapentin (50 microM and 100 microM), reported to impede calcium channels, was ineffective. Inhibition of low threshold T-type calcium channels by mibefradil (10 microM) significantly reduced potassium (by 47%) but not capsaicin-stimulated iCGRP release. Reduction of total sodium channel conductance by tetrodotoxin (1 microM), lidocaine (10 microM, 50 microM or 500 microM) or by replacement of extracellular sodium with choline-chloride did not result in a reduction of either potassium- or capsaicin-induced axonal iCGRP release. These results suggest that slow depolarization by high extracellular potassium activates axonal low threshold (T-type) as well as high threshold-activated (L-type) voltage-gated calcium channels to mediate iCGRP release, and that capsaicin-induced release is largely dependent on calcium influx through TRPV1. Action potential generation and propagation are not required for axonal release mechanisms.

Morphological characterization of rat Mas-related G-protein-coupled receptor C and functional analysis of agonists.

A recently described family of "orphan" receptors, called Mas-related G-protein-coupled receptors (Mrg), is preferentially expressed in small nociceptive neurons of the rodent and human dorsal root ganglia (DRG). Mrg are activated by high affinity peptide fragments derived from the proenkephalin A gene, e.g. BAM22 (bovine adrenal medullary). To study the histological distribution and functional properties of these receptors, we combined confocal immunohistochemistry in rat DRG and dermis whole mounts, using new antibodies against the rat Mas-related G-protein-coupled receptor C (MrgC), with single-fiber recordings and neurochemical experiments using isolated hind-paw skin and sciatic nerve. In lumbar DRG we found cytoplasmic MrgC labeling mainly in small- and medium-sized neurons; coexpression with isolectin B4 (46%) and transient receptor potential vanilloid receptor 1 channel protein (TRPV1) (52%) occurred frequently, whereas calcitonin gene-related peptide (CGRP) was rarely colocalized with MrgC in DRG (11%) and dermal nerve fibers (6%). One of the MrgC agonists, BAM22, more than doubled the heat-induced cutaneous CGRP release from rat and mouse skin. The effect of BAM22, also known to activate opioid receptors, was further enhanced by combination with naloxone that had no effect on its own. This sensitizing effect proved to be independent of secondary prostaglandin formation, mast cell degranulation, protein kinase C (PKC) activation and independent of TRPV1. Nonetheless, the capsaicin-induced CGRP release was also sensitized. Receptive fields of 26 mechano-heat sensitive C-fibers were treated with MrgC agonists. Only one unit was strongly and repeatedly excited and showed a profound sensitization to heat upon BAM22+naloxone. Two other established MrgC agonists (gamma2-melanocyte stimulating hormone and BAM8-22) were ineffective. Thus, BAM22 sensitizes the capsaicin- and heat-induced CGRP release in an apparently MrgC-unrelated way. The sensitization to heat appears unusually resistant against pharmacological interventions and does not involve TRPV1.

Electrophysiological characterization of vagal afferents relevant to mucosal nociception in the rat upper oesophagus.

Emerging evidence indicates a nociceptive role of vagal afferents. A distinct oesophageal innervation in the rat, with muscular and mucosal afferents travelling predominantly in the recurrent (RLN) and superior laryngeal nerve (SLN), respectively, enabled characterization of mucosal afferents with nociceptive properties, using novel isolated oesophagus-nerve preparations. SLN and RLN single-fibre recordings identified 55 and 14 units, respectively, with none conducting faster than 8.7 m s(-1). Mucosal response characteristics in the SLN distinguished mechanosensors (n = 13), mechanosensors with heat sensitivity (18) from those with cold sensitivity (19) and a mechanoinsensitive group (5). The mechanosensitive fibres, all slowly adapting, showed a unimodal distribution of mechanical thresholds (1.4-128 mN, peak approximately 5.7 mN). No difference in response characteristics of C and Adelta fibres was encountered. Mucosal proton stimulation (pH 5.4 for 3 min), mimicking gastro-oesophageal reflux disease (GORD), revealed in 31% of units a desensitizing response that peaked around 20 s and faded within 60 s. Cold stimulation (15 degrees C) was proportionally encoded but the response showed slow adaptation. In contrast, the noxious heat (48 degrees C) response showed no obvious adaptation with discharge rates reflecting the temperature's time course. Polymodal (69%) mucosal units, > 30% proton sensitive, were found in each fibre category and were considered nociceptors; they are tentatively attributed to vagal nerve endings type I, IV and V, previously morphologically described. All receptive fields were mapped and the distribution indicates that the posterior upper oesophagus may serve as a 'cutbank', detecting noxious matters, ingested or regurgitated, and triggering nocifensive reflexes such as bronchoconstriction in GORD.

The TRPV1/2/3 activator 2-aminoethoxydiphenyl borate sensitizes native nociceptive neurons to heat in wildtype but not TRPV1 deficient mice.

TRPV1 gene disruption results in a loss of capsaicin and proton responsiveness, but has minimal effects on heat-induced nocifensive behavior, suggesting that sensory transduction of heat is independent of TRPV1. TRPV3, another heat-activated ion channel but insensitive to capsaicin, was shown to be expressed in keratinocytes as well as in sensory neurons projecting to the skin. Recently, 2-aminoethoxydiphenyl borate was introduced as a TRPV3 agonist, but its selectivity was questioned by showing that it activated recombinant TRPV1 and TRPV2 as well. We used the isolated mouse skin-saphenous nerve preparation and whole-cell patch-clamping of cultured dorsal root ganglia neurons from TRPV1-/- and wildtype mice. We found no phenotypic differences between the heat responses of polymodal C-fibers, whereas cultured dorsal root ganglia neurons of TRPV1-/- hardly showed any heat-activated currents. Only C-fibers of wildtype but not TRPV1-/- mice were clearly sensitized to heat by 2-aminoethoxydiphenyl borate 10 and 100 microM; heat-activated current in wildtype neurons was only facilitated at 100 microM. Noxious heat-induced calcitonin gene-related peptide release showed clear deficits (<50%) in TRPV1 deficient skin, but the stimulated calcitonin gene-related peptide release from the isolated skull dura was unaffected. In both models, 2-aminoethoxydiphenyl borate was able to potentiate the heat response (46 degrees C, 5 min) in a concentration-dependent manner, again, only in wildtype but not TRPV1-/- mice, suggesting that TRPV2/3 are not involved in this sensitization to heat. The results further suggest that TRPV1 is not responsible for the normal heat response of native nociceptors but plays the essential role in thermal sensitization and a prominent one in controlling dermal calcitonin gene-related peptide release, i.e. neurogenic inflammation.

Can receptor potentials be detected with threshold tracking in rat cutaneous nociceptive terminals?

Threshold tracking of individual polymodal C- and Adelta-fiber terminals was used to assess membrane potential changes induced by de- or hyperpolarizing stimuli in the isolated rat skin-nerve preparation. Constant current pulses were delivered (1 Hz) through a tungsten microelectrode inserted in the receptive field, and the current amplitude was controlled by feedback with a laboratory computer programmed to serially determine the electrical threshold using the method of limits. During threshold tracking, the receptive fields of the fibers were heated (32-46 degrees C in 210 s) or superfused with modified synthetic interstitial fluid containing either 0, 20, 40, 50, or 60 mM [K+], phosphate buffer to pH 5.2 or 6.1, or bradykinin (BK, 10(-8)-10(-5) M). High [K+]e decreased the current threshold for activation by 6-14% over 120 s, whereas K+-free superfusion augmented the threshold by >5%, and after some delay, also induced ongoing discharge in 60% of units. pH 6.1 and 5.2 caused an increase in threshold of 6 and 18%, respectively, and 30% of the fibers were excited by low pH, although the change in threshold of pH responsive and unresponsive fibers did not differ significantly, suggesting a general excitability decrease induced by protons. Heat stimulation increased the mean threshold and conduction velocity of the fibers tested and resulted in activity in 78% of units. Additionally, for these units, activation was preceded by a significant decrease in threshold compared with the tracked thresholds of fibers unresponsive to heat. Bradykinin also led to a significant threshold decrease before activation. In conclusion, the technique of threshold tracking proved suitable to assess changes in excitability resulting from receptor currents evoked by noxious heat and bradykinin in the terminal arborization of cutaneous nociceptors.

Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse.

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles--presumably on axonal transport--as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10(-6) M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures--the omega structures--lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

Responsiveness of C-fiber nociceptors to punctate force-controlled stimuli in isolated rat skin: lack of modulation by inflammatory mediators and flurbiprofen.

Although cutaneous C-fiber nociceptors show dramatic inflammatory sensitization to heat, they do not appear to get sensitized to the mechanical stimulation by von Frey hairs. We employed force-controlled punctate electromechanical stimulation to receptive fields of 61 characterized C-fibers in the isolated rat skin-saphenous nerve preparation. In general: low-in contrast to higher-threshold units showed greater dynamic sensitivity and response magnitude, an earlier onset and a stronger degree of adaptation, the latter due to the linear rise of the force stimulus. On this methodological basis three groups of units were subject to a mix of inflammatory mediators, to flurbiprofen or to control solution. Subsequent mechanostimulation revealed a good reproducibility of the control response and no significant changes in the treatment groups. In conclusion, even refined mechanostimulation was unable to demonstrate sensitization of the predominant nociceptor classes in the rat skin.

Injection pain of rocuronium and vecuronium is evoked by direct activation of nociceptive nerve endings.

BACKGROUND AND OBJECTIVE: Rocuronium and, to a lesser extent, vecuronium can induce burning sensations associated with withdrawal reactions during administration. Dermal microdialysis in human and electrophysiological recordings of nociceptors in mouse skin were used to elucidate the underlying mechanisms of pain induction. METHODS: Microdialysis catheters were inserted intradermally into the forearm of 10 volunteers and were perfused with two different concentrations of rocuronium and vecuronium (1 and 10 mg mL(-1)) or a control. Dialysis samples were taken every 15 min and analysed for protein, histamine, tryptase and bradykinin content. Pain intensity was rated on a numerical scale of 0-10. In a parallel design, activation of cutaneous nociceptors was assessed directly in a skin-nerve in vitro preparation of the mouse hind paw. The receptive fields of identified single C-nociceptors (n = 12) were superfused with rocuronium or vecuronium solutions (10 mg mL(-1)) at physiological pH. RESULTS: In accordance with clinical observations, microdialysis of rocuronium (10 mg mL(-1)) induced sharp burning pain (NRS 4.1 +/- 1.8), whereas vecuronium given in the usual clinical concentration (1 mg mL(-1)) induced only minor pain sensations (NRS 0.6 +/- 1.3). At equimolar concentrations, pain sensation and concomitant mediator release evoked by both drugs were similar. No correlations were found between pain rating and mediator release. In the in vitro preparation, C-fibres showed a consistent excitatory response with rapid onset after stimulation with vecuronium as well as rocuronium (differences not significant). CONCLUSIONS: The algogenic effect of aminosteroidal neuromuscular blocking drugs can be attributed to a direct activation of C-nociceptors.