Voltage-dependent Ca2+ currents in rat cardiac dorsal root ganglion neurons

This study presents the kinetic and pharmacological properties of voltage-gated Ca(2+) currents in anatomically defined cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of fluorescent tracer Fast Blue into the pericardial sack. There were three distinct groups of neurons with respect to cell size: small (27% of total; cell capacitance <30 pF), medium (65% of total; capacitance 30-80 pF) and large neurons (8% of total; capacitance >80 pF). The properties of Ca(2+) currents were tested in small and medium-sized neurons. In large neurons currents could not be adequately controlled and were not analysed. Ca(2+) currents did not completely inactivate during 100 ms depolarising voltage steps. The activation thresholds in small (-36.9+/-1.3 mV) and medium (-39.0+/-1.3 mV) size neurons were similar. Current densities were 105.8 pA/pF in small and 97.4 pA/pF in large neurons and also did not differ. The kinetic properties of activation and inactivation did not differ between small and medium-sized cardiac DRG neurons. At membrane potentials between -50 and -60 mV (the expected resting membrane potential in these neurons) 55 to 70% of Ca(2+) currents in small and medium-sized neurons were available for activation. Both, small and medium-sized neurons expressed similar proportions of L (7.5%), N (25%) and P/Q (36%) type Ca(2+) currents. We conclude that small and medium-sized cardiac DRG neurons are homogeneous with respect to the expression and properties of voltage-gated Ca(2+) currents. Voltage-gated Ca(2+) currents probably play an important role in action potential generation in cardiac DRG neurons due to their availability for activation at resting membrane potential, their high density and voltage threshold close to the threshold for voltage-gated Na(+) currents.     

Phenylarsine oxide as a redox modulator of transient receptor potential vanilloid type 1 channel function

Transient receptor potential vanilloid type 1 (TRPV1) channels are capable of detecting and integrating noxious stimuli and play an important role in nociceptor activation and sensitization. It has been demonstrated that oxidizing agents are capable of positively modulating (sensitizing) the TRPV1 channel. The present study investigates the ability of the thiol‐oxidizing agent phenylarsine oxide (PAO) to modulate TRPV1 currents under voltage‐clamp conditions. We assessed the ability of PAO to modulate both proton‐ and capsaicin‐activated currents mediated by recombinant human TRPV1 channels as well as native rat and human TRPV1 channels in dorsal root ganglion (DRG) neurons. Experiments with other oxidizing and reducing agents having various membrane‐permeating properties supported the intracellular oxidizing mechanism of PAO modulation. The PAO modulation of proton‐activated currents was consistent across the cell types studied, with an increase in current across the proton concentrations studied. PAO modulation of the capsaicin‐activated current in hTRPV1/Chinese hamster ovary cells consisted of potentiation of the current elicited with low capsaicin concentrations and inhibition of the current at higher concentrations. This same effect was seen with these recombinant cells in calcium imaging experiments and with native TRPV1 channels in rat DRG neurons. Contrary to this, currents in human DRG neurons were potentiated at all capsaicin concentrations tested after PAO treatment. These results could indicate important differences in the reduction–oxidation modulation of human TRPV1 channels in a native cellular environment. © 2014 Wiley Periodicals, Inc.     

Vanilloid receptor TRPV1 is not activated by vanilloids applied intracellularly

The vanilloid receptor TRPV1 is a ligand-gated cation channel that can be activated by capsaicin, acids and noxious heat. For vanilloids, a stretch of approximately 8 amino acids in the vicinity of the TM3 region on the cytosolic side of TRPV1 and R114 and E761 in the N- and C-cytosolic tails, respectively, has been shown to be critical for capsaicin binding and channel activation. Here, we report that intracellular application of vanilloids is insufficient for activating TRPV1 channels in HEK293T cells. Pipette solution (ICS) for recording membrane currents was supplemented with 50 microM capsaicin (n=14) or 1 microM resiniferatoxin (RTX) (n=39) and the responses induced by extracellular capsaicin (1 microM) or RTX (100 nM) were recorded at intervals >50% of that needed for diffusion of Lucifer yellow from the pipette to reach maximum fluorescence (n=7). We found that all cells with expressed TRPV1 exhibited a similar sensitivity to vanilloids irrespective of whether the membrane currents were recorded with electrodes filled with ICS containing capsaicin or RTX or only with control ICS. We suggest that, in addition to intracellularly located agonist recognition sites of TRPV1, there is at least one resides on the extracellular side, which needs to be occupied to activate the channel.     

Cellular mechanism of action of resiniferatoxin: a potent sensory neuron excitotoxin

The mechanism of activation of sensory neurons by the potent irritant resiniferatoxin (RTX) was compared with that of the pungent compound, capsaicin. RTX and capsaicin evoked an inward, depolarising current associated with an increase in membrane conductance in a subpopulation of dissociated cultured neurons from rat dorsal root ganglia. RTX also evoked an uptake of⁴⁵Ca into and an efflux of [¹⁴C]guanidinium and of⁸⁶Rb from these cells but was at least 100-fold potent than capsaicin. The levels of cGMP, but not cAMP were elevated by RTX. Prolonged exposure to RTX damaged DRG neurons by a predominantly osmotic process. RTX-sensitive cells were identified by a cobalt-staining method; neurofilament-containing DRG neurons were RTX-insensitive as were all sympathetic neurons and non-neuronal cells. Cultured DRG neurons from chick embryos were also unaffected by RTX. In a neonatal rat spinal cord-tail preparation in vitro, RTX activated capsaicin-sensitive peripheral noiciceptive fibres and caused a subsequent spinal cord depolarization measured in the ventral spinal roots. Neither prolonged exposure to a phorbol ester, to desensitize/down-regulate protein kinase C, nor inhibition of protein kinase C by staurosporine affected responses produced by RTX or capsaicin. The effects of capsaicin were abolished when preparations were exposed to desensitizing concentrations of RTX. RTX therefore acts as a highly potent capsaicin analogue to activate a subpopulation of rat sensory neurons.     

Activation of transient receptor potential vanilloid 2‐expressing primary afferents stimulates synaptic transmission in the deep dorsal horn of the rat spinal cord and elicits mechanical hyperalgesia

Probenecid, an agonist of transient receptor vanilloid (TRPV) type 2, was used to evaluate the effects of TRPV2 activation on excitatory and inhibitory synaptic transmission in the dorsal horn (DH) of the rat spinal cord and on nociceptive reflexes induced by thermal heat and mechanical stimuli. The effects of probenecid were compared with those of capsaicin, a TRPV1 agonist. Calcium imaging experiments on rat dorsal root ganglion (DRG) and DH cultures indicated that functional TRPV2 and TRPV1 were expressed by essentially non‐overlapping subpopulations of DRG neurons, but were absent from DH neurons and DH and DRG glial cells. Pretreatment of DRG cultures with small interfering RNAs against TRPV2 suppressed the responses to probenecid. Patch‐clamp recordings from spinal cord slices showed that probenecid and capsaicin increased the frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents in a subset of laminae III–V neurons. In contrast to capsaicin, probenecid failed to stimulate synaptic transmission in lamina II. Intrathecal or intraplantar injections of probenecid induced mechanical hyperalgesia/allodynia without affecting nociceptive heat responses. Capsaicin induced both mechanical hyperalgesia/allodynia and heat hyperalgesia. Activation of TRPV1 or TRPV2 in distinct sets of primary afferents increased the sEPSC frequencies in a largely common population of DH neurons in laminae III–V, and might underlie the development of mechanical hypersensitivity following probenecid or capsaicin treatment. However, only TRPV1‐expressing afferents facilitated excitatory and/or inhibitory transmission in a subpopulation of lamina II neurons, and this phenomenon might be correlated with the induction of thermal heat hyperalgesia.     

Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat

The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52 degrees C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Adelta and Abeta fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Abeta fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Abeta afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents-in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing.     

Pharmacological characterization of vanilloid receptor located in the brain

Specific [3H]resiniferatoxin (RTX) binding detects the vanilloid receptor type I (VR1). In the present study we demonstrate specific, high-affinity, saturable [3H]RTX binding in various areas of monkey brain not known to be innervated by primary afferent neurons as well as in spinal cord and dorsal root ganglion neurons of the same origin. Detailed pharmacological characterization and comparison revealed no major difference in binding affinities between the peripheral and the central sites as measured by K(d)/K(i) values. In general, lower receptor density was measured in selected brain areas than in the periphery. Areas with higher receptor density were detected in the locus ceruleus, preoptic area, and medial basal hypothalamus of the brain. Both capsaicin and the competitive antagonist capsazepine inhibited the specific binding of [3H]RTX to membrane preparations of the dorsal horn of the spinal cord and dorsal root ganglia with K(i) values of 4.3+/-0.32 microM and 2.7+/-0.33 microM, respectively. Inhibition was observed in the central areas (hypothalamus) with K(i) values of 0.95+/-0.1 microM for capsaicin and 0.86+/-0.11 microM for capsazepine. Previous biological and pharmacological evidence suggested that vanilloid receptors were present in the brain. Our results demonstrate that the pharmacological properties of both the peripheral and central receptor sites display appropriate pharmacological similarity to represent the same receptor class. The modest differences in ligand affinities for the vanilloid receptor expressed in the brain nuclei and the dorsal root ganglion neurons may correspond to differences in sequence, modification or associated proteins.     

Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

The [³H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [³H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [³H]RTX bound with similar affinity and positive cooperatively to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 ± 3.5 pM, 38.4 ± 3.1 pM and 42.6 ± 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 ± 0.11, 1.78 ± 0.12 and 1.78 ± 0.09, respectively. The maximal binding capacity was 0.218 ± 0.026 fmol/10³ cells and 0.196 ± 0.021 fmol/10³ cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [³H]RTX from cells in suspension with K_i and Hill coefficient values of 42.5 ± 5.3 pM, 2.06 ± 0.16 μM, 3.16 ± 0.21 μM and 32.4 ± 4.1 nM and 1.79 ± 0.17, 1.68 ± 0.06, 1.72 ± 0.11 and 1.81 ± 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing ⁴⁵Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of ⁴⁵Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 ± 2.9 pM, 25.7 ± 3.0 nM, 68.5 ± 3.8 nM, 173 ± 25 pM, 7.98 ± 0.83 μM and 4.93 ± 0.35 μM as compared to 0.94 ± 0.12 nM, 26.5 ± 3.5 nM, 149 ± 30 nM, 1.46 ± 0.25 nM, 1.41 ± 0.48 μM and 340 ± 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperatively of receptor binding; however, stimulation of ⁴⁵Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [³H]RTX by rat dorsal root ganglion membrane preparations with K_i and Hill coefficient values of 3.89 ± 0.38 μM and 1.74 ± 0.11. On the other hand it inhibited the induction of ⁴⁵Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with K_i values of 271 ± 29 nM and 325 ± 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperatively. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of ⁴⁵Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.     

Spontaneous neuronal activity in organotypic cultures of mouse dorsal root ganglion leads to upregulation of calcium channel expression on remote Schwann cells

It is well established that neurons regulate the properties of both central and peripheral glial cells. Some of these neuro-glial interactions are modulated by the pattern of neuronal electrical activity. In the present work, we asked whether blocking the electrical activity of dorsal root ganglion (DRG) neurons in vitro by a chronic treatment with tetrodotoxin (TTX) would modulate the expression of the T-type Ca(2+) channel by mouse Schwann cells. When recorded in their culture medium, about one-half of the DRG neurons spontaneously fired action potentials (APs). Treatment for 4 days with 1 microM TTX abolished both spontaneous and evoked APs in DRG neurons and in parallel significantly reduced the percentage of Schwann cells expressing Ca(2+) channel currents. On the fraction of Schwann cells still expressing Ca(2+) channel currents, these currents had electrophysiological parameters (mean amplitude, mean inactivation time constant, steady-state inactivation curve) similar to those of control cultures. Co-treatment for 4 days with 1 microM TTX and 2 mM CPT-cAMP, a cAMP analogue that induces the expression de novo of Ca(2+) channel currents in Schwann cells deprived of neurons, maintained the percentage of Schwann cells expressing Ca(2+) channel currents, showing that TTX does not directly affect the expression of Ca(2+) channel currents by Schwann cell. We conclude that blocking spontaneous activity of DRG neurons in vitro downregulates Ca(2+) channel expression by Schwann cells. These results strongly suggest that DRG neurons upregulate Ca(2+) channel expression by Schwann cells via the release of a diffusible factor whose secretion is dependent on electrical activity.     

Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglion membranes

We have previously demonstrated that resiniferatoxin (RTX), an unusual phorbol-related diterpene, induces similar responses in rodents to those induced by capsaicin, the pungent constituent of hot peppers (the genus Capsicum). Strikingly, RTX was 3-4 orders of magnitude more potent than was capsaicin. We report here specific binding of [3H]RTX to particulate preparations from dorsal root ganglia (DRG), a target tissue of both RTX and capsaicin action. The Kd was 0.27 nM for DRG from the rat; the Bmax was 160 fmol/mg. The respective values for pig DRG were 2.2 nM and 730 fmol/mg. Typical phorbol esters did not inhibit [3H]RTX binding. Capsaicin inhibited binding with 10(4)-fold lower affinity than RTX, consistent with the relative in vivo potencies. The specific [3H]RTX binding appears to represent the postulated capsaicin receptor.     

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (I(Ca(V))). I(Ca(V)) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba2+ as charge carrier. In large diameter neurones (>35 micorm), a concentration of 50 microM was needed to reduce I(Ca(V)) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (< or =30 microm), the IC50 was 0.36 microM. This effect was concentration dependent with a threshold below 0.025 microM and maximal blockade (>80%) at 5 microM. The current-voltage relation was shifted to the hyperpolarized direction with an increase of the current between -40 and -10 mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 microM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I(Ca(V)) at negative and decreases I(Ca(V)) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.     

Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain

Protons cause a sustained depolarization of human dorsal root ganglion (DRG) neurons [Baumann et al. (1996) Pain, 65, 31-38]. In the present study we sought to determine which ion channels are expressed in human DRG neurons that could mediate the sustained responses observed in the patch-clamp recordings. RT-PCR of material from the DRG tissue revealed the presence of mRNAs for a nonselective cation channel that is activated by protons (TRPV1) and background potassium channels that are blocked by protons (TASK-1, TASK-3 and Kir2.3). Highly acidic solution (pH 5.4) applied to cultured DRG neurons evoked prolonged currents that were associated with a net increase in membrane conductance. Consistent with the involvement of TRPV1, these proton-evoked currents were blocked by capsazepine and were only found in neurons that responded to capsaicin with an increase in membrane conductance. Less acidic extracellular solution (pH 6.0) evoked such currents only rarely, but was able to strongly enhance the currents evoked by capsaicin. Capsazepine (1 microm) blocked the currents evoked by capsaicin at pH 7.35, as well as the potentiated responses to capsaicin at pH 6.0. In neurons that were not excited by capsaicin, moderate extracellular acidification (pH 6.0) caused a sustained decrease in resting membrane conductance. The decrease in membrane conductance by protons was associated with inhibition of background potassium channels. This excitatory effect of protons was not blocked by capsazepine. We conclude that in most neurons the sustained depolarization in response to moderately acidic solutions is the result of blocked background potassium channels. In a subset of neurons, TRPV1 also contributes.     

Modulation of voltage-activated Ca currents by pain-inducing agents in a dorsal root ganglion neuronal line,F-11

Whole cell currents evoked by pain-inducing agents—bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (I_Na) was 100 μA/cm² and the I_Na was abolished by 10^?6 M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient I_Ca(t) and a high threshold sustained I_Ca(I). In addition, another high threshold transient Ca current, presumably I_Ca(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of ?70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10^?6 M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10^?6 M Bk in the majority of the cells. Similarly, both 10^?6 M Cap and 10^?9 M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (G_i). © 1993 Wiley-Liss, Inc.     

Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy.

Sensitivity to the pungent vanilloid, capsaicin, defines a subpopulation of primary sensory neurons that are mainly polymodal nociceptors. The recently cloned vanilloid receptor subtype 1 (VR1) is activated by capsaicin and noxious heat. Using combined in situ hybridization and histochemical methods, we have characterized in sensory ganglia the expression of VR1 mRNA. We show that this receptor is almost exclusively expressed by neurofilament-negative small- and medium-sized dorsal root ganglion cells. Within this population, VR1 mRNA is detected at widely varying levels in both the NGF receptor (trkA)-positive, peptide-producing cells that elicit neurogenic inflammation and the functionally less characterized glial cell line-derived neurotrophic factor-responsive cells that bind lectin Griffonia simplicifolia isolectin B4 (IB4). Cells without detectable levels of VR1 mRNA are found in both classes. A subpopulation of the IB4-binding cells that produce somatostatin has relatively low levels of VR1 mRNA. A previously uncharacterized population of very small cells that express the receptor tyrosine kinase (RET) and that do not label for trkA or IB4-binding has the highest relative levels of VR1 mRNA. The majority of small visceral sensory neurons of the nodose ganglion also express VR1 mRNA, in conjunction with the BDNF receptor trkB but not trkA. Axotomy results in the downregulation of VR1 mRNA in dorsal root ganglion cells. Our data emphasize the heterogeneity of VR1 mRNA expression by subclasses of small sensory neurons, and this may result in their differential sensitivity to chemical and noxious heat stimuli. Our results also indicate that peripherally derived trophic factors may regulate levels of VR1 mRNA.     

Effects of systemic resiniferatoxin treatment on substance P mRNA in rat dorsal root ganglia and substance P receptor mRNA in the spinal dorsal horn

Capsaicin depletes the sensory neuropeptide substance P (SP) in the rat due to a combination of neuron loss and decreased synthesis in the surviving cells. Resiniferatoxin (RTX) mimics most, but not all, capsaicin actions. In the present study, the effects of RTX (300 μg/kg, s.c.) were examined on mRNA levels for SP and its receptor in the adult rat. The percentage of dorsal root ganglia (DRG) neuronal profiles showing an in situ hybridization signal for preprotachykinin mRNAs encoding SP was not altered following RTX treatment (up to 8 weeks), though the signal became perceptibly weaker. In accord, 2 weeks after RTX administration a 60% decrease was observed in the steady-state levels of SP-encoding mRNAs using Northern blot analysis, leaving the ratio of β- and γ-preprotachykinin mRNAs unchanged. No change was, however, observed in mRNA levels encoding tachykinins NK-1 receptors in the dorsal horn, the spinal targets for SP. The present findings suggest that RTX does not kill SP-positive DRG neurons, though it suppresses the synthesis of SP. Since RTX treatment does not alter NK-1 receptor expression, this reduced SP synthesis is likely to play a central role in the analgesic actions of RTX.     

Effect of Surgical and Chemical Sensory Denervation on Non-neural Expression of the Transient Receptor Potential Vanilloid 1 (TRPV1) Receptors in the Rat

Pretreatment with the ultrapotent capsaicin analog resiniferatoxin (RTX) has been applied as a selective pharmacological tool in inflammation and pain studies to desensitize transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerve endings. The discovery of TRPV1 receptor on non-neural cells challenges systemic RTX desensitization as a method acting exclusively on a population of sensory neurons, but not on non-neural cells. Systemic RTX desensitization was used for chemical denervation and transection of the sciatic and saphenous nerves for surgical denervation in rats. Quantitative real-time PCR and immunohistochemistry were applied to investigate the presence and alterations of the TRPV1 receptor mRNA and protein following chemical and surgical denervation. We provided the first evidence for non-neural TRPV1 immunopositivity and mRNA expression in the rat dorsal paw and plantar skin as well as the oral mucosa. Neither chemical nor surgical denervation influenced the level of TRPV1 receptor mRNA and protein expression in non-neural cells of either skin regions or mucosa. Therefore, RTX and consequently capsaicin remain to be considered as selective neurotoxins for a population of primary afferent neurons.     

Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root anglion membranes

We have previously demonstrated that resiniferatoxin (RTX), an unusual phorbol-related diterpene, induces similar responses in rodents to those induced by capsaicin, the pungent constituent of hot peppers (the genus Capsicum). Strikingly, RTX was 3–4 orders of magnitude more potent than was capsaicin. We report here specific binding of [³H]RTX to particulate preparations from dorsal root ganglia (DRG), a target tissue of both RTX and capsaicin action. The K_d was 0.27 nM for DRG from the rat; the B_max was 160 fmol/mg. The respective values for pig DRG were 2.2 nM and 730 fmol/mg. Typical phorbol esters did not inhibit [³H]RTX binding. Capsaicin inhibited binding with 10⁴-fold lower affinity than RTX, consistent with the relative in vivo potencies. The specific [³H]RTX binding appears to represent the postulated capsaicin receptor.     

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用(英文)

目的观察神经生长因子(nerve growth factor, NGF)对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中P物质(substance P, SP)的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体(vanilloid receptor 1, VR1)mRNA的表达,用放射免疫分析(radioimmunoassay,RIA)法检测SP的基础释放量和辣椒素(100 nmol/L)刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。