Sensitization of Cutaneous, Neuronal Purinergic Receptors Contributes to Endothelin-1-Induced Mechanical Hypersensitivity

Abstract

Endothelin (ET-1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre-exposure of ND7/104 immortalized sensory neurons to ET-1 (30 nM) for 10 min increased the proportion of cells responding to ATP (2 μM) with an increase in intracellular calcium, an effect prevented by the ET_A receptor-selective antagonist BQ-123. ET-1 (3 nM) pre-exposure also increased the proportion of isolated mouse DRG neurons responding to ATP (0.2-0.4 μM). Blocking ET-1-evoked increases in intracellular calcium with the IP₃ receptor antagonist 2-APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET-1-mediated intracellular calcium increases. ET-1-sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using qPCR and receptor-selective ligands in ND7/104 showed that ET-1-induced sensitization most likely involves the P2X4 receptor subtype. ET-1-sensitized calcium responses to ATP were strongly inhibited by broad spectrum (TNP-ATP) and P2X4-selective (5-BDBD) antagonists, but not antagonists for other P2X subtypes. TNP-ATP and 5-BDBD also significantly inhibited ET-1-induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET-1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors.

Introduction

Endothelin-1 (ET-1) is a potent vaso-active peptide that also plays a prominent role in peripheral pain, but its mechanisms are complex and incompletely understood. ET-1 acts through two G-protein coupled receptors (ET_A and ET_B) on neuronal and non-neuronal cells in the skin to mediate both pro- and anti-nociceptive effects [3; 17]. When injected into the rat plantar hind paw, ET-1 at high doses causes overt pain (as indicated by robust hind paw flinching) and sensitization to thermal and mechanical stimulation. Lower doses of ET-1 are also capable of producing tactile sensitization, but do not cause overt pain [12]. The overt pain elicited by subcutaneous ET-1 injection into the paw has been almost exclusively attributed to the direct activation of ET_A receptors on nociceptive sensory neurons that innervate the skin, since these nerves express ET_A receptors and increase their firing in vivo in response to ET-1 administration [12; 28]. ET_A receptor activation also results in enhanced excitability in the soma of isolated nociceptive primary sensory neurons, through alterations in ionic currents and sensitization of excitatory receptors. Activation of ET_A receptors promotes TTX-sensitive sodium currents, reduces delayed rectifier potassium currents and sensitizes TRPV1 receptors, all processes which likely contribute to tactile sensitization following ET-1 administration [10; 24; 32; 35].

Recently, our lab has shown that endogenous release of excitatory molecules in the skin appears to contribute to ET-1-induced mechanical sensitization. Pre-injection of antagonists for NMDA glutamate receptors reduced both the early and late phases of this sensitization, while an antagonist of the calcitonin gene-related peptide (CGRP) receptor reduced only the late phase [18]. ET-1 was found to increase the release of both of these molecules from cultured dorsal root ganglion (DRG) neurons through ET_A receptor activation, suggesting that ET-1 injection into the skin causes mechanical allodynia in part by enhancing the release of glutamate and CGRP from cutaneous nerve terminals.

In addition to causing increased release of algogenic substances in the skin, it is also possible that ET-1 sensitizes ligand-gated receptors on nociceptive nerve terminals. ET_A receptor-expressing nociceptive neurites terminate in the epidermis where they are surrounded by keratinocytes. Keratinocytes release a variety of pro-algesic molecules that activate excitatory receptors on nociceptive nerve endings, including glutamate, CGRP and ATP [2; 11; 15; 20; 33]. In particular, cutaneous ATP release has been implicated in numerous types of acute and chronic pain [5; 9]. Subcutaneously administered purinergic receptor antagonists are effective analgesics in a variety of animal pain models, including ones of inflammatory and neuropathic pain [6; 22; 30]. For this study, we hypothesized that ET-1-induced mechanical hypersensitivity is partly due to sensitization of purinergic receptors expressed by primary sensory neurons. We provide evidence that ET_A receptor activation enhances ATP responses in cultured sensory neurons independent of resulting intracellular calcium increases and that cutaneous ATP release in the skin contributes to ET-1-induced mechanical hypersensitivity in the rat hind paw.

Materials and Methods

ND7/104 cell culture

ND7/104 model sensory neurons, a cell line derived from embryonic rat DRG neurons hybridized with mouse neuroblastoma N18GT2 cells, were generously donated in 2004 by Dr. P. Hogan (Harvard Medical School, Boston, MA). These cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with penicillin and streptomycin (100 μg) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C and 5% CO₂. For calcium imaging, cells were plated onto poly-L-lysine-coated cover slips so that they were approximately 50-80% confluent when imaging was performed the next day.

Isolation and culture of mouse sensory neurons (mDRG neurons)

Male adult CD1 mice (Charles River, Wilmington, MA) were purchased and housed in the animal facilities of Children’s Hospital Boston on a 12-hour alternating light-dark cycle. Mice were experimentally treated and cared for using policies and procedures approved by the Harvard Committee on Animals and conformed to the guidelines of the Committee for Research and Ethical Issues of IASP. Animals for imaging were dissected after 7 weeks of age. After CO₂ asphyxiation and cervical translocation, and following spinal laminectomy, the left and right DRG from the whole spine were removed and placed in 4°C Hanks-buffered saline solution (HBSS, Life Technologies, Grand Island, NY). After the DRG were collected and centrifuged for 3 minutes at 1000 rpm (150 × g), they were placed in a collagenase/dispase solution (3 mg/mL dispase II and 1 mg/mL collagenase A, Roche Applied Science) and incubated at 37°C for 90 minutes. After incubation, the cells were washed in DMEM (Life Technologies), fortified with 4.5 g/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate, 10% fetal bovine serum (Life Technologies), penicillin (500 U/mL, Cellgro, Manassas, VA), and streptomycin (500 μg/mL, Cellgro). DNAse (125 U/mL, Sigma) was then added and the solution was triturated using successively smaller caliber, flame-polished Pasteur pipettes. This solution was gently layered onto a bovine serum albumin gradient (10% albumin from bovine serum, Sigma in PBS, Life Technologies) and spun at 150 × g for 12 minutes to reduce the proportion of satellite cells. After removal of the supernatant, the cells were washed again in DMEM, suspended in neurobasal medium (Life Technologies) supplemented with L-glutamine (20 mM, Life Technologies), B-27 supplement (Life Technologies), penicillin (500 U/mL, Cellgro), streptomycin (500 μg/mL, Cellgro), nerve growth factor (NGF; 50 ng/mL, Life Technologies), glial derived nerve factor (GDNF; 2 ng/mL, Sigma), and arabinocytidine (Ara-C; 10 μM, Sigma) and plated onto poly-D-lysine (100 mg/mL, Sigma) and laminin-treated (1 mg/mL, Sigma) round glass cover slips (Fisher Scientific), and then maintained in an incubator at 37°C (5% CO₂). Media was completely replaced 2 days post-dissection, and all imaging was performed four days after dissection and culture. Preliminary studies showed that both ET-1 and ATP responses were most robust four days after isolation (data not shown). Cultures of cells isolated from mouse DRG contained both primary sensory neurons and satellite cells. In calcium imaging experiments, neurons were distinguished based on their increase in intracellular calcium in response to 40 mM potassium chloride (KCl).

qPCR

RNA was isolated from 3 separate passages of 80% confluent ND7/104 cultures using an RNeasy kit (Qiagen, Valencia, CA) according to manufacturer's instructions. The concentration and quality of RNA were measured using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE), after which 1 μg of total RNA was synthesized to cDNA using an iScript kit (Biorad, Hercules, CA), according to manufacturer's instructions. Samples were also prepared substituting reverse transcriptase with RNAse-free water to control for contamination. qPCR was conducted using Evagreen^TM PCR supermix on a miniopticon thermocycler (Biorad, Hercules, CA) using the following protocol: 94°C for 3 min, 45 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, and then a melting curve (65°C to 95°C in 0.5°C increments) to confirm primer specificity. Two separate qPCR experiments were each conducted in duplicate on the three ND7/104 samples for a total of four replicates per data point.

Sequences of the primers used to amplify P2X receptors have been previously published [21]. Data were analyzed using BioRad CFX manager. Expression of each target was normalized to the housekeeping gene Cyclo-A, adjusting for primer efficiencies calculated using REST 2009 software (Qiagen) from a serial dilution of each primer.

Calcium imaging of cells

Calcium imaging experiments were conducted largely as previously described [23]. Intracellular calcium was determined by excitation microfluorometry using the Ca⁺⁺-sensitive fluorescent dye, fura-2AM (Invitrogen). Coverslips were first rinsed with Ringer's solution (NaCl 155 mM, KCl 4.5 mM, CaCl₂ 2 mM, MgCl₂ 1 mM, D-glucose 10 mM, HEPES 5 mM, pH 7.4) and incubated in fura-2AM (4 μM) in Ringer's for 30 min in the dark at room temperature. The cover slip was then transferred to an imaging chamber and rinsed with Ringer's solution to remove excess dye. Using a pipette, 1 mL volumes of test solutions were added to an approximately 200 μL pool covering the cells, with the chamber's volume maintained by a vacuum removal line. Cells were monitored using an inverted IX17 microscope (Olympus America Inc., PA) equipped with a Lambda DG-4+ digital camera (Hamamatsu Photonics, Japan). Two alternating excitation wavelengths, 340/380 nm, were provided by a Xenon Arc Bulb (Sutter Instrument, CA) and the emitted light passed through a 510 nm interference filter to a photomultiplier tube. Intracellular calcium levels were determined by the ratio of fura-2 fluorescence using the following equation:

$$\frac{\Delta F}{F_0}=\frac{(F-F_0)}{F_0}$$

where F is the fluorescence light intensity at each time point and F₀ is the baseline fluorescence intensity averaged over 10 sec before the stimulus application. Responding cells were defined as those with >10% increase in ΔF/F₀. Data analysis focused on changes in the percent of cells responding to a treatment, due to variability in the amplitude of ET-1-mediated changes in the calcium response to ATP (data not shown). For simplicity, the proportion of cells responding to a stimulus will be referred to as the "calcium response" to that stimulus. Thus, an “increase in ATP calcium response” following ET-1 treatment refers to an increase in the % of cells responding to ATP after ET-1 pre-treatment.

Effect of ET-1 pre-treatment on ATP responses

In order to determine the effect of ET-1 on ATP responses in ND7/104 cells, fura signals were measured in response to a variety of ATP concentrations, with and without ET-1 pre-treatment. Cells were pre-treated with Ringer's (control) or ET-1 (30 nM) for 10 min followed by exposure to ATP at different concentrations (vehicle, 500 nM, 2 μM, 8 μM and 16 μM). Responses to ATP were recorded until all cells’ fluorescence returned to approximately baseline levels (5-10 min depending on ATP concentration). Each coverslip was used for only a single concentration of ATP. Data were analyzed with a 2-way factorial ANOVA with post-hoc t-tests to compare ATP responses between ET-1 pre-treated and vehicle pre-treated groups for each concentration of ATP. Graphpad Prism (La Jolla, CA) was used to calculate the EC50 for ATP with and without ET-1 pre-treatment using a non-linear regression function agonist concentration-response curve with the maximum constrained at 100%.

mDRG neurons were more responsive to both ET-1 and ATP compared to ND7/104 cells. In contrast to experiments on ND7/104 cells, where ET-1 was present until the addition of ATP, ET-1 had to be rinsed away within 5-10 sec using three 1 mL rinses of Ringer's solution for intracellular calcium levels to return to baseline. Responses in mDRG could also vary between isolations, thus preliminary experiments were conducted on each batch to find the concentration of ET-1 and ATP that caused a response in approximately 50% of cells (an approximate EC50). In determining the effect of ET-1 on ATP responses in mDRG neurons, two batches of mDRG neurons (8 coverslips/batch) were tested. In the first batch, 4 coverslips were treated with ET-1 (6 nM) or vehicle followed by 3 × 1 mL rinses of Ringer's within 5-10 sec. Ten minutes later, cells were treated with ATP (200 nM) for 5 min and then KCl (40 mM) for 2 min. In the second batch of mDRG, another 8 coverslips were treated the same, except using ET-1 and ATP concentrations of 3 nM and 400 nM, respectively. Data from both batches were combined for statistical analysis and analyzed with independent sample t-tests.

Identification of receptors and role for calcium in ET-1-mediated sensitization

To determine which ET receptor subtype was involved in the sensitization of ND7/104 to ATP, the ET_A receptor-selective antagonist BQ-123 and the ET_B receptor-selective antagonist BQ-788, were used. Coverslips were pre-treated with BQ-123 (100 nM) or BQ-788 (100 nM) for 5 min before addition of the same antagonist alone or in combination with ET-1 (30 nM) for 10 min. Subsequently, ATP (2μM) was added for 5 min in the absence of antagonists. Data were analyzed with planned comparison t-tests to determine whether the antagonists inhibited ET-1 evoked calcium responses (antagonist alone vs. antagonist with ET-1) and/or sensitization of ATP calcium responses (ATP after antagonist treatment alone vs. ATP after antagonist with ET-1).

To confirm involvement of ATP receptors in the sensitized response to ATP, the broad spectrum purinergic receptor antagonist TNP-ATP was tested in ND7/104 and mDRG cultures. For ND7/104 cultures, TNP-ATP (100 μM) or vehicle was added for 5 min, followed by ET-1 (30 nM) with or without TNP-ATP for 10 min and then ATP with or without TNP-ATP (4 μM) for 5 min. In mDRG cultures, ET-1 (3 nM) was added and rinsed away as described above. On the third rinse, 100 μM TNP-ATP (or vehicle control) was added for 10 min, followed by ATP (400 nM) with or without TNP-ATP for 3 min and then KCl (40 mM) alone for 2 min.

Ca⁺⁺-free Ringer's (NaCl 155 mM, KCl 4.5 mM, MgCl₂ 3 mM, D-glucose 10 mM, HEPES 5 mM, pH 7.4) was used first to determine whether the source of ATP-mediated increases in intracellular calcium was extracellular. After loading of fura-2AM in standard Ringer's, coverslips were placed in the recording chamber and rinsed 3 times with 1 mL of Ca⁺⁺-free or standard (2 mM Ca⁺⁺) Ringer's and all subsequent reagents were added in Ringer's with the appropriate [Ca⁺⁺]. For ND7/104 cultures, cells were incubated in Ringer's for 5 min, then ET-1 (30 nM) for 10 min, followed by ATP (2 μM) for 5 min. For mDRG neurons, cells were incubated in Ringer's for 5 min, then ET-1 (3 nM) for 10 min with 3 × 1 mL rinses within 5-10 sec, followed by ATP (1 μM) for 3 min. KCl could not be used to distinguish neuronal cells in these experiments, due to the absence of calcium in the media. Thus, data regarding the effect of Ca⁺⁺-free Ringer's include both DRG neurons and satellite cells.

Since experiments with Ca⁺⁺-free Ringer's indicated ionotropic P2X receptor involvement in the sensitized ATP response, a combination of P2X receptor-selective ligands was used to identify which of the expressed P2X subtype(s) mediated the sensitized ATP response in ND7/104. P2X ligands were chosen based on qPCR results for P2X subtypes expressed by ND7/104 cultures and included the P2X3 receptor-selective antagonist A317491 (1 μM), the P2X4 receptor-selective antagonist 5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD, 1 μM), the P2X4 positive allosteric modulator ivermectin (10 μM), the P2X7 receptor-selective antagonist brilliant blue G (BBG, 1 μM), and the P2X2 partial agonist P¹,P⁴-Di(adenosine-5′) tetraphosphate ammonium (Ap4a, 100 μM). Ap4a, BBG, A317491 and ivermectin were purchased from Sigma, while TNP-ATP and 5-BDBD was purchased from Tocris (Bristol, UK). A low calcium Ringer's solution (NaCl 155 mM, KCl 4.5 mM, CaCl₂ 0.3 mM, MgCl₂ 2.7 mM, D-glucose 10 mM, HEPES 5 mM) was used for P2X receptor antagonist and ivermectin experiments.

For antagonist experiments, cells were pre-treated for 5 min with the appropriate antagonist or low calcium Ringer's alone (control), followed by ET-1 (30 nM) for 10 min, then ATP (4 μM) for 5 min. Data from P2X receptor antagonist experiments were analyzed using independent sample t-tests to compare the percent of cells that responded to ATP after sensitization by ET-1 in the presence or absence of the P2X antagonist.

Experiments using Ca⁺⁺-free Ringer's, purinergic receptor antagonists and ivermectin were conducted with their own individual control groups using the appropriate vehicle. Experiments alternated between control and antagonist treatments to limit inter-experiment variability. For simplicity, a single control group is displayed in results, with error bars representing the average SEM of all control groups (Figure 3B and 5A). Calculation of percent control and statistical analyses were done by comparing each treatment to its own control group.

Figure 3. Characterization of the purinergic receptor sensitized by ET-1.

A. Pharmacological characterization of the P2X receptors involved in the calcium response to ATP following ET-1 sensitization by ET-1. Effects of P2X receptor antagonists were measured on ATP responses following ET-1 sensitization, as the purinergic receptors mediating the calcium response to ATP could be different following sensitization. Bars represent % cells responding to ATP following ET-1 (30 nM for 10 min) pre-treatment and under the conditions noted in the X axis. Each treatment was normalized to its own individual control group (dotted line). Compared to standard (2 mM Ca⁺⁺) Ringer's, low calcium Ringer's (0.3mM) reduced, and calcium-free Ringer's (0 mM Ca⁺⁺) completely abolished the responses to ATP (4 μM) after pre-exposure to ET-1. The broad spectrum purinergic receptor antagonist TNP-ATP and the P2X4 receptor-selective antagonist 5-BDBD both significantly reduced ET-1-sensitized ATP calcium responses. Ivermectin, a P2X4 specific potentiator, significantly increased cells responding to ATP (with no ET-1 pre-treatment). The P2X3 and P2X7 receptor-selective antagonists A317491 and brilliant blue G were without effect. B. qPCR for P2X receptor expression in ND7/104. qPCR was used to identify P2X receptor subtypes present in cultures. Transcripts for P2X2, 3, 4 and 7 were highly expressed (mean +/− SEM, n = 3). C. There is currently no selective antagonist available for the P2X2 receptor, thus the P2X2 partial agonist Ap4a was used to determine whether ET-1 sensitized this subtype. Cells were pretreated with ET-1 (30 nM) or Ringer's alone for 10 min (Treatment 1), followed by Ap4a (100 μM) or Ringer's alone for 5 min (Treatment 2) and then ATP (2 μM, Treatment 3). Significantly more cells responded to ET-1 compared to Ringer's, but no increase in responses was seen for Ap4a. ET-1 also did not sensitize cells to Ap4a, but did increase ATP calcium responses. Data in A and C are mean +/− SEM for n = 6-8 (n = 3 for 0.3 mM Ca⁺²), *p < 0.05, ns = not significant.

Figure 5. P2X receptor-mediated calcium responses are sensitized by ET-1 in mouse dorsal root ganglion (mDRG) neurons, independent of ET-1-evoked increases in intracellular calcium.

A. Quantification of calcium imaging of mDRG neuron responses to ATP. Pre-treatment with ET-1 for 10 min significantly increased the proportion of cells responding to ATP compared to pre-treatment with Ringer's (Control). Values are mean +/− SEM with n = 8 (ET-1 and Control) or n = 4 (TNP-ATP and 0 mM Ca⁺⁺). Each treatment was normalized to its own individual control group. For simplicity, only the control for ET-1 alone is shown. Administration of the broad-spectrum purinergic receptor antagonist TNP-ATP after ET-1 pre-treatment, but 10 min before ATP administration, strongly reduced ATP calcium responses. When cells were pre-treated with ET-1 for 10 min and then exposed to ATP in the absence of extracellular calcium (0 mM Ca⁺⁺), calcium responses to ATP were largely absent. B. As in ND7/104, 2-APB alone caused a gradual, low level increase in intracellular calcium in mDRG neurons. 2-APB completely prevented ET-1-evoked increases in intracellular calcium, but did not significantly reduce the ability of ET-1 to sensitize ATP calcium responses. Values are mean +/− SEM with n = 5. C. Correlation between peak calcium responses of mDRG neurons to ET-1 and ATP. As in ND7/104, peak levels of intracellular calcium evoked by ET-1 were poorly correlated with the same cell’s subsequent increase in intracellular calcium caused by ATP (r² = 0.02). Each point represents a single cell. *p < 0.05, ns = not significant (p > 0.05).

In ivermectin experiments, cells were pre-treated with ivermectin or low calcium Ringer's alone (control) for 5 min and then ATP (4 μM) for 5 min. The percent of cells responding to ATP with and without ivermectin pre-treatment were compared using independent sample t-tests. For experiments testing the P2X2 partial agonist Ap4a, cells were treated with Ringer's alone or ET-1 (30 nM) for 10 min followed by Ap4a for 5 min and then ATP (4 μM) for 5 min. Data from Ap4a experiments were measured using oneway ANOVA with Tukey's post-hoc tests.

Preliminary experiments showed that some purinergic receptor ligands could not be used in calcium imaging due to non-specific effects. PPADS inhibited ND7/104 responses to ET-1 and suramin, NF110, NF279 and phenolphthalein caused increases in intracellular calcium themselves and/or interfered with the fura-2AM fluorescence.

To assess the dependence of ATP sensitization on the increase in intracellular calcium caused by ET-1, the IP₃ receptor antagonist 2-Aminoethoxydiphenylborane (2-APB, Tocris) was used. For ND7/104 cultures, 2-APB (50 μM) was added for 10 min, followed by 2-APB alone or in combination with ET-1 (30 nM) for 10 min and then by ATP (4 μM) for 5 min. In mDRG cultures, 2-APB (10 μM) was added for 10 min, followed by 2-APB alone or in combination with ET-1 (3 nM) for 10 min and then by ATP (1 μM) for 3 min and KCl (40 mM) for 2 min. Data from 2-APB experiments were analyzed using independent sample t-tests.

Behavioral Studies

Animals were experimentally treated and cared for using policies and procedures approved by the Harvard Committee on Animals and conformed to the guidelines of the Committee for Research and Ethical Issues of IASP. Experiments were performed on adult male Sprague-Dawley rats (240-330 g, Charles River, USA), housed 2 per cage under a 12:12 hour dark:light cycle and provided with food and water ad libitum.

Injection procedures

ET-1 (AXXORA, LLC, San Diego, CA) was dissolved in PBS (pH=7.4; Invitrogen) at a concentration 0.1 mg/0.1 ml and stored in aliquots for up to 2 weeks at −80°C. Stock solution was diluted in PBS to its final concentration. During all experiments, the working solutions were kept on ice.

The broad spectrum purinergic receptor antagonist TNP-ATP and the P2X4 receptor-selective antagonist 5-BDBD were used in behavioral experiments to determine whether endogenous ATP release plays a role in ET-1-induced mechanical allodynia. Rats received ET-1 alone (with appropriate antagonist vehicle, as described below), ET-1 + TNP-ATP, or ET-1 + 5-BDBD. For TNP-ATP treated rats, TNP-ATP (10 mM, 10 μl) was injected subcutaneously into the plantar hind paw 25 min before and then co-injected at 5 mM concentration with 10 μM ET-1. For co-injections, a manufactured stock water solution of TNP-ATP (10 mM) was mixed V:V with 20 μM ET-1. For 5-BDBD treated rats, 5-BDBD (10 mM, 10 μl) was pre-injected 25 min before, but not co-injected with ET-1, due to differences in solubility. In vehicle controls for TNP-ATP, an injection of 10 μl of H₂O was followed by an injection of 20 μM ET-1 alone mixed V:V with H₂O. In vehicle controls for 5-BDBD, an injection of 10 μl of DMSO (the vehicle for 5-BDBD) was followed by an injection of 10 μM ET-1 alone in H₂O. TNP-ATP and 5-BDBD were also tested in the absence of ET-1 to control for any effects on mechanical sensitivity of these drugs alone.

Test solutions were injected into the mid-plantar hind paw, 1 cm distal from the heel using a 30-G needle attached to a 10 μl Hamilton microsyringe (Hamilton Co., Reno, NV, USA). Injection occurred under brief general anesthesia (0.5-1 min), which was accomplished with the rapidly reversible, inhalational agent sevoflurane (Abbott Labs, N. Chicago, IL). Anesthesia was evident by the animal’s flaccid paralysis, usually occurring within 10-15 sec, and recovery of the righting reflex occurred < 30 sec after the anesthetic-containing tube was removed from the face.

Mechanical testing

Unrestrained rats were placed on an elevated plastic mesh floor (28 × 17.5 cm; 9.5 × 9.5 mm openings) and allowed to habituate for 25-40 min before baseline testing was initiated. Paw Withdrawal Frequency (PWF) to mechanical stimulation was determined starting at 30 minutes post-injection (enough time for any residual effects of anesthesia to have dissipated) using calibrated von Frey hairs (VFH) applied perpendicular to the plantar surface of a hind paw through spacing in the mesh. Each VFH (4 g, 10 g and 15 g) was applied 10 times, for 3 sec each time, with a 3 sec interval. Testing with the next VFH started ca. 8-10 min after the beginning of testing with the previous, lower force. Testing started with the lowest force of 4 g, and continued with increasing forces, with all three forces tested with 10 probings in each test period. To avoid stress and to get consistent responsiveness to the same force, the rats were habituated by handling and tested on mesh racks over 5-6 days before each experiment (training period). The number of paw withdrawals (range: 0-10) occurring in response to 10 trials was used as the quantitative measure of sensitivity, and graphed as PWF for each force.

Since behavioral data were not normally distributed, non-parametric statistics were used for these analyses. For each treatment (ET-1 alone and ET-1 + TNP-ATP or ET-1 + 5-BDBD), Friedman's test was used to determine whether there was a difference in PWF across all time points, followed by Dunn's post-hoc tests to identify those time points when PWF significantly differed from baseline. Mann-Whitney tests were also performed to determine whether PWFs were significantly different between groups of rats receiving ET-1 alone and either ET-1 + TNP-ATP or ET-1 + 5-BDBD, for the individual forces at specified time points. Friedman’s test was also used to determine whether PWF was significantly different from baseline following TNP-ATP or 5-BDBD treatment alone.

Software used for statistical analyses

Data are presented as means ± S.E.M and evaluated using Statistica version 6.0 (in vitro experiments; Statsoft, Tulsa, OK) or GraphPadInStat version 3.0 (in vivo experiments; GraphPad Software, CA, USA).

Results

Effect of ET-1 on ATP responses in ND7/104

Pre-treatment with ET-1 increased the proportion of ND7/104 cells responding to ATP with an increase in intracellular calcium (Figure 1). Overall, addition of ET-1 (30 nM) alone caused calcium responses in 39.33 ± 9.53% of cells (n = 34), significantly higher than a rinse with Ringer's alone, which caused calcium responses in 2.64 ± 1.61% of cells (t-test p < 0.001, data not shown, but see examples in figure 1C and D). Ten minutes after ET-1 exposure, a sufficient time for intracellular calcium levels to return close to baseline, ATP was added (Figure 1D). The increase in the number of ATP-responding cells as a function of [ATP] is shown in Figure 2A. Significant main effects were found for both pre-treatment (p < 0.01) and ATP concentration (p < 0.001), along with a significant interaction (p < 0.05). ET-1 pre-treatment significantly increased calcium responses to 2 μM ATP (p < 0.05), without significantly affecting the response to Ringer's alone (0.61 ± 0.39% responding for control vs. 3.48 ± 1.41% responding for ET-1, p = 0.08). Exposure to ET-1 shifted the EC50 for ATP from 2.85 ± 1.23 μM to 1.50 ± 1.36 μM (Figure 2A).

Figure 1. ET-1 pre-treatment sensitizes ND7/104 cells to ATP.

Images and traces from representative experiments showing changes in intracellular calcium in vehicle (A, C) and ET-1 (B, D) pre-treated ND7/104 visualized by fura calcium imaging. Images show intracellular calcium levels at the times indicated (in sec). Arrows mark the times that test solutions were added. Each trace shows the change in intracellular calcium levels of a single cell relative to baseline. Cells were exposed to ET-1 (30 nM) or Ringer’s solution alone (vehicle) for 10 min, followed by addition of 2 μM ATP. Increases in intracellular calcium can be seen in response to both ET-1 and ATP, but calcium responses occur very infrequently in vehicle treated cells.

Figure 2. ET-1 sensitizes ND7/104 to ATP through ET_A receptor activation.

A. Concentration-response curve of calcium imaging data for the percent of cells responding to ATP with (squares) and without ET-1 (circles) pre-treatment. Cells were treated with Ringer's alone (control) or ET-1 (30 nM) for 10 min and then with ATP at varying concentrations. ET-1 exposure significantly increased the proportion of cells responding to ATP at 2 μM. B. Endothelin receptor dependence of ATP sensitization. Cells were pre-incubated for 5 min (Treatment 1) with the ET_A receptor-selective antagonist BQ-123 (100 nM) or the ET_B receptor-selective antagonist BQ-788 (100 nM) which were then added in combination with ET-1 (30 nM) or vehicle for 10 min (Treatment 2) followed by ATP alone (2 μM) for 5 min (Treatment 3). ET-1 failed to cause calcium responses in the presence of BQ-123, but was still able to in the presence of BQ-788. Furthermore, pre-incubation with BQ-123, but not BQ-788, prevented the ET-1-mediated increase in ATP responsive cells. Graphs represent mean +/− SEM with n = 6-9. *p < 0.05, ns = not significant.

Effect of endothelin receptor antagonists on ET-1 sensitization of ATP responses in ND7/104

The ET_A receptor-selective antagonist BQ-123 and the ET_B receptor-selective antagonist BQ-788 were used to determine which endothelin receptor was responsible for sensitization to ATP (Figure 2B). BQ-123, added 5 min before and together with ET-1, prevented any significant increase in ET-1 calcium responses, above that of BQ-123 alone (4.48 ± 1.91% responding for BQ-123 alone vs. 5.80 ± 1.43% responding for BQ-123 with ET-1, t-test p = 0.59). In contrast, BQ-788 failed to prevent ET-1 calcium responses (p < 0.05). Importantly, BQ-123 (p = 0.49), but not BQ-788 (p < 0.001), also prevented any increase in ATP calcium responses after treatment with ET-1, compared to when the antagonist was given alone (Figure 2B).

Characterization of the purinergic receptor involvement in the sensitized ATP response in ND7/104

Calcium imaging was conducted in ND7/104 cultures using reduced calcium (0.3 mM and 0 mM) Ringer's solution and selective ligands for P2X receptor subtypes (Figure 3A). As has been previously published by our lab [34], the absence of extracellular calcium did not prevent ND7/104 cells from responding to ET-1 (data not shown, p < 0.001). The absence of extracellular calcium did, however, prevent any significant increase in ATP calcium responses compared to Ca⁺⁺-free Ringer's alone (Figure 3A, p = 0.58). Reducing extracellular calcium from 2 mM to 0.3 mM also reduced the percent of cells responding to ATP, although this did not reach statistical significance with the low number of coverslips analyzed (p = 0.054, n = 3).

qPCR was conducted on ND7/104 cultures using specific primers for mouse P2X receptor subtypes. qPCR analysis showed that transcripts for P2X2, P2X3, P2X4 and P2X7 were the most highly expressed, with very low levels of P2X1, P2X5 and P2X6 detected (Figure 3B). All products yielded single peak melt curves and substitution of reverse transcriptase with nuclease-free water during cDNA generation prevented the detection of any amplification products following qPCR. Since ND7/104 cells are a hybrid of mouse and rat cells, qPCR results were confirmed using specific primers for rat P2X receptor subtypes, which showed similar results (data not shown).

Next, ligands selective for the P2X receptor subtypes identified by qPCR were tested to determine which ones contributed to the sensitized ATP response (Figure 3A). The ability of antagonists to reduce the sensitized ATP response was assessed using independent sample t tests, comparing the proportion of cells responding to 2 μM ATP after ET-1 pre-treatment in the presence and absence of the antagonist. The broad spectrum purinergic receptor antagonist TNP-ATP, added 5 min before and together with ET-1, significantly inhibited ET-1 sensitized ATP calcium responses (p < 0.01), as did the P2X4 selective antagonist 5-BDBD (p < 0.05). Furthermore, the P2X4 receptor-specific potentiator ivermectin significantly increased calcium responses to 4 μM ATP, by 4-fold (Figure 3A, p < 0.05) in experiments using no ET-1 pre-treatment. A317491 (p=0.83) and brilliant blue G (BBG, p = 0.57), selective antagonists for P2X3 and P2X7 receptors, respectively, were without effect. None of the P2X-selective ligands used significantly altered the percent of cells responding to ET-1 or vehicle pretreatment (data not shown). The P2X2 selective partial agonist Ap4a failed to cause calcium responses above that seen for Ringer's alone, even with ET-1 pre-treatment (Figure 3C, p = 0.35 and 0.76). ET-1 was still able to sensitize ATP calcium responses in these same cells (p < 0.01).

Test for the role of calcium in the sensitization mechanism in ND7/104

Calcium imaging was conducted using the IP₃ receptor antagonist 2-APB (Figure 4). 2-APB (50 μM) itself caused a small, gradual increase in intracellular calcium in ND7/104 (possibly due to TrpV receptor activation [7]), with 22.2 ± 5.71% of cells showing an increase over 10% from baseline. Addition of 2-APB 10 min before and then together with ET-1 prevented any ET-1-mediated calcium increases above 2-APB alone (p = 0.052), although a small number of cells still showed low level responses to ET-1 (Figure 4B). In cells treated with 2-APB alone, only 2.71 ± 1.41% of cells responded to 4 μM ATP. In contrast, coverslips pretreated with ET-1 in the presence of 2-APB showed responses to 4 μM ATP in 33.12 ± 8.72% of cells, significantly higher than 2-APB alone (Figure 4C, p < 0.01). Correlations were also calculated to determine the relationship between the peak calcium responses to ET-1 and those to ATP (Figure 4D). For all concentrations of ATP tested, there was no relationship between the peak increase in intracellular calcium caused by ET-1 pre-treatment compared to the peak intracellular calcium increase caused by ATP measured in the same cell (r² = 0.01). In fact, many cells responded well to ATP, after little or no response to ET-1. When the responses to each concentration of ATP (500 nM, 2 μM, 8 μM and 16 μM) were compared individually, correlation coefficients were still extremely low (r² = 0.02-0.10, data not shown).

Figure 4. Calcium independence of ET-1 sensitization of ATP responses in is not dependent on ET-1-mediated increases in intracellular calcium.

A and B. Representative traces of ND7/104 pre-incubated with the IP₃ receptor antagonist 2-APB (50 μM) for 5 min (Treatment 1) followed by ET-1 (30 nM) with 2-APB or 2-APB alone for 10 min (Treatment 2) and then ATP (4 μM) for 5 min (Treatment 3). Arrows illustrate the addition of test media and each trace represents a single cell. C. Quantification of the 2-APB effect on ET-1 sensitization. 2-APB caused a gradual, low level increase in intracellular calcium. In the presence of 2-APB, ET-1 failed to cause calcium responses above that of 2-APB alone. Despite pre-incubation with 2-APB, ET-1 exposure was still able to sensitize ATP calcium responses. Values are mean +/− SEM with n = 5-9. *p < 0.05, ns = not significant (p > 0.05). D. Correlation between peak calcium responses of cells to ET-1 (30 nM) and ATP (500 nM-16 μM). Data from the concentration-response curve (figure 2A) was used to measure the correlation between the peak calcium response elicited from ET-1 pre-treatment with the subsequent peak calcium response to ATP in the same cell. The level of intracellular calcium caused by ET-1 showed no relationship to the same cell’s subsequent increase in intracellular calcium caused by ATP (r² = 0.01). Each point represents a single cell.

Effect of ET-1 on ATP responses in mDRG neurons

In mDRG neurons, pre-treatment with ET-1 (3 or 6 nM) for 10 min significantly increased calcium responses to ATP (200 or 400 nM) compared to pre-treatment with Ringer's alone (Figure 5A). As in Figure 3A, data in Figure 5A are shown normalized to control and each treatment was normalized to its own individual control group. ET-1 caused calcium responses in 43.68 ± 5.53% of cells, significantly higher than a rinse with Ringer's alone which caused calcium responses in 7.75 ± 3.20% of cells (t-test p < 0.001, data not shown). ET-1 pre-treatment led to a response to ATP in 56.60 ± 7.52% of cells, significantly higher than with Ringer's pre-treatment (35.01 ± 5.35% responding, t-test p = 0.031).

Characterization of purinergic receptor involvement in the sensitized ATP response in mDRG neurons

Calcium imaging was conducted using Ca⁺⁺-free Ringer's and TNP-ATP to confirm a P2X receptor-mediated response in mDRG neurons (Figure 5A). The absence of extracellular calcium did not significantly affect ET-1 calcium responses (p = 0.38), but strongly reduced ATP calcium responses after ET-1 sensitization (p < 0.001). TNP-ATP also significantly reduced ATP calcium responses after ET-1 pre-treatment (Figure 5A, p < 0.01), while lacking any significant effect on the percent of KCl responders (data not shown, p = 0.76).

Test for the role of calcium in the sensitization mechanism in mDRG neurons

2-APB also caused small, gradual calcium responses in mDRG neurons, while preventing intracellular calcium increases in response to ET-1 (Figure 5B, p = 0.75). 2-APB had no effect on KCl calcium responses (data not shown, p = 0.68). Pre-treatment with ET-1 in the presence of 2-APB still increased ATP calcium responses, compared with those pre-treated with 2-APB alone (p < 0.01). In addition, no correlation was seen between peak calcium responses to ET-1 and peak calcium responses to ATP in mDRG neurons (r² = 0.02, Figure 5C).

Effect of TNP-ATP and 5-BDBD on ET-1-induced mechanical hypersensitivity

Paw withdrawals to tactile stimulation by 4 g, 10 g and 15 g von Frey filaments were measured 30 min to 4 hours after injection of ET-1 alone or with TNP-ATP or 5-BDBD (Figure 6A-C). Injection of ET-1 alone caused a significant increase in PWF above baseline to all three forces (Friedman's test, p < 0.05). PWF was significantly increased from baseline at 30 min for all three forces (Dunn's test p < 0.05 for 4 g and 10 g, p < 0.001 for 15 g), as well as for the 10 g force at 3 hours post-ET-1 injection (Dunn's test p < 0.05).

Figure 6. Inhibition of purinergic signaling reduces ET-1-induced mechanical hypersensitivity in the rat hind paw.

Effects of the broad spectrum purinergic receptor antagonist TNP-ATP and the P2X4 receptor-selective antagonist 5-BDBD were measured on ET-1-induced mechanical hypersensitivity. TNP-ATP or vehicle (for ET-1 alone group) was pre-injected (10 mM) 25 min before and then co-injected (5 mM) with ET-1 (10 μM) subcutaneously into the hind paw. 5-BDBD (10mM) or vehicle (for ET-1 alone group) was pre-injected 25 min before ET-1 (10 μM). Rats were tested for mechanical sensitivity using 4 g (A, D), 10 g (B, E), and 15 g (C, F) von Frey filaments beginning 30 min after injection. The graph shows mechanical sensitivity as measured by paw withdrawal frequency (PWF, the number of times out of 10 trials that a paw withdrawal occurred in response to the designated von Frey force). Effect of TNP-ATP (A-C). ET-1 alone significantly increased PWF above baseline at 30 min post-injection for all forces tested, as well as at 3 hours post-injection for the 10 g force (*). When TNP-ATP was co-administered, ET-1 only caused a significant increase in PWF above baseline for the 15 g force at 30 min after injection. Furthermore, TNP-ATP treated rats showed significantly lower PWF in response to the 10 g force at 1 and 3 hours post-injection compared to ET-1 alone (X). Effect of 5-BDBD (D-F). Pre-treatment with 5-BDBD prevented any increases in PWF above baseline, except for the 10 g force at 30 min post-injection. In contrast, ET-1 alone caused significant increases from baseline for the 10 g and 15 g forces at 30 min and 1 hour post-injection (*). 5-BDBD significantly reduced PWF evoked by ET-1 for the 10 g and 15 g forces at 30 min post-injection (X). Data are mean +/− SEM for n = 9-11. *p < 0.05 vs. baseline (Dunn's test), ^Xp < 0.05 ET-1 vs. ET-1 with antagonist (Mann-Whitney test). Controls for antagonist effects on mechanical sensitivity (G-I). TNP-ATP or 5-BDBD was injected as described above in the absence of ET-1 and PWF was recorded beginning 30 min post-injection. Neither antagonist caused a significant change in PWF from baseline for any force measured. Data are mean +/− SEM for n = 6.

Pre-injection of TNP-ATP reduced the hypersensitivity from ET-1. When TNP-ATP was co-injected with ET-1, there was still an overall significant increase in PWF above baseline for the 4 g and 15 g forces (Friedman's test p < 0.05), but not for the 10 g force (Friedman's test p = 0.08). Despite a significant main effect of time in the ET-1 + TNP-ATP treated group for the 4 g force, none of the individual time points reached a significant increase in PWF above baseline by Dunn's post-hoc tests. For the 15 g force, only the 30 min time point showed significantly higher PWF over baseline (Dunn's test p < 0.05). TNP-ATP treated rats showed significantly lower PWF to the 10 g force at 1 and 3 hours post-injection compared to rats treated with ET-1 alone (Mann-Whitney p < 0.05).

Pre-injection of 5-BDBD also significantly reduced hypersensitivity compared to that seen from ET-1 alone (Figure 6D-F). ET-1 alone caused a significant increase in PWF above baseline for the 10 g and 15 g forces at 30 min and 1 hour post-injection (Friedman's test p < 0.05, followed by Dunn's post-hoc tests p < 0.05). When 5-BDBD was pre-injected before ET-1, PWF was only significantly increased above baseline for the 10 g force 30 min after injection (Friedman's test p < 0.05, followed by Dunn's post-hoc tests p < 0.05). 5-BDBD-treated rats showed significantly lower PWF to the 10 g and 15 g forces at 30 min and 1 hour post-injection compared to rats treated with ET-1 alone (Mann Whitney p < 0.05). Neither TNP-ATP nor 5-BDBD had any significant effect on PWF in the absence of ET-1 for any force tested (Friedman’s test p = 0.145-0.976, Figure 6G-I).

Discussion

ET_A receptor activation sensitizes ND-7/104 and mDRG cells to ATP, independent of ET-1 mediated increases in intracellular calcium

Pre-treatment with ET-1 increased the proportion of ND-7/104 cells (Figure 1 and 2A) and isolated mDRG neurons (Figure 5A) responding to ATP. This sensitization appears to be independent of the rise in intracellular calcium elicited by ET-1, as blocking this intracellular calcium increase with 2-APB failed to prevent sensitization of ATP calcium responses (Figure 4 and 5B). In fact, the sensitizing effect of ET-1 was even more dramatic than in the absence of 2-APB for both ND7/104 and mDRG neurons. Furthermore, no correlation was seen between the peak calcium amplitude elicited by ET-1 (derived from intracellular stores) and the subsequent peak calcium amplitude in response to ATP (from the extracellular solution) in the same cell (Figure 4D and 5C). It thus appears that changes in intracellular calcium resulting from ET-1 cannot account for ET-1’s potentiation of the response to ATP.

While it has been demonstrated that P2X receptors can be sensitized by stimuli such as low pH and inflammatory mediators, the intracellular signaling mechanisms that underlie this sensitization remain largely undetermined [13; 26; 31]. More research will be needed to define the intracellular pathway that underlies ET-1 sensitization of purinergic receptor signaling, which may involve a Gα_q-triggered mechanism unrelated to IP₃-stimulated calcium release or, possibly, ET_A receptor coupling to other Gα or Gβγ subunits [17; 23].

We have previously reported that ND-7/104 cells express both ET_A and ET_B receptors, with ET_A being more highly expressed and the dominant receptor mediating ET-1-evoked increases in intracellular calcium [23]. Unlike ND7/104 cells, primary sensory neurons in vivo appear to exclusively express ET_A receptors and the pro-nociceptive effects of cutaneous ET-1 injection into the rat hind paw (flinching and mechanical hyperalgesia) are ET_A receptor mediated [8; 10; 12; 28; 35]. Importantly, the sensitizing effect of ET-1 on ATP responses in ND7/104 is mediated through ET_A receptors. Pre-incubation with the ET_A receptor-selective antagonist BQ-123 abolished both the direct calcium response to ET-1 itself, as well as the sensitization of the ATP response (Figure 2B).

ET-1 sensitizes P2X responses in ND7/104 and mDRG neurons

Replacing most or all calcium with magnesium in the test media, respectively, reduced or abolished the calcium response of ND7/104 to ATP following ET-1 pre-treatment, indicating that the receptor activated by this concentration of ATP was an ionotropic P2X receptor subtype (Figure 3B). Similarly, ET-1 sensitized calcium responses to ATP in mDRG cultures were mostly absent in the presence of Ca⁺⁺-free media (Figure 5A). The lack of extracellular calcium prevented the use of KCl to distinguish neuronal cells from satellite cells in these experiments, but the effect of calcium removal on almost all cells indicates P2X-mediated responses to 400 nM ATP in both cell types. Removal of calcium from the media did not reduce responses to ET-1, which is known to cause increases in cytoplasmic calcium through IP₃-mediated release from intracellular stores [23].

Data from calcium imaging experiments using P2X receptor-selective ligands indicate that ET-1 acts through ET_A to sensitize an ATP response in ND7/104 cells that is likely mediated largely through the P2X4 receptor subtype (Figure 3A). The broad spectrum purinergic receptor antagonist TNP-ATP significantly inhibited ET-1-sensitized ATP calcium responses in ND7/104, confirming involvement of purinergic receptors. P2X4 is highly expressed in ND7 and the P2X4 receptor-selective antagonist 5-BDBD significantly reduced the percent of cells responding to ATP following sensitization by ET-1. In addition, the P2X4 receptor-specific potentiator ivermectin significantly enhanced the proportion of cells responding to ATP at the dose sensitized by ET-1, while antagonists for P2X3 and P2X7 had no effect. The most pronounced ET-1 sensitization in ND7/104 was to 2 μM ATP, a dose consistent with activation of P2X4 [16]. Despite the presence of P2X2 mRNA, the P2X2 receptor-selective partial agonist Ap4a failed to elicit any detectable increases in intracellular calcium, even when used after ET-1 pre-treatment and at almost 7 times the reported EC50 [27].

The strong inhibitory effects of TNP-ATP and removal of extracellular calcium on ET-1-sensitized ATP responses in mDRG strongly indicate the involvement of P2X receptors in these cells. Although the specific P2X receptor subtype(s) underlying mDRG responses to ATP were not identified, P2X4 receptors are widely expressed in trigeminal and dorsal root ganglion sensory neurons, including TrkA-expressing nociceptive neurons. Over 80% of rat lumbar DRG neurons express P2X4, half of which also co-express the TrkA receptor [19]. Although much research has focused on the role for glial P2X4 receptors in pain mechanisms, this widespread expression of P2X4 in nociceptive DRG neurons suggests that neuronal P2X4 receptors may also play a role in pain [29].

Endogenous release of excitatory molecules in the skin contributes to ET-1 nociceptive responses

As previously demonstrated in our lab, local subcutaneous injection of ET-1 caused mechanical hypersensitivity of the injected rat hind paw (Figure 6) [1; 18]. TNP-ATP and 5-BDBD, the broad-spectrum and P2X4-selective purinergic receptor antagonists that inhibited ET-1-sensitized ATP responses in ND7/104 and mDRG neurons in culture, also reduced the mechanical hypersensitivity induced by ET-1 injection (Figure 6). Co-administration of TNP-ATP limited the ET-1 induced increase in PWF in response to all forces tested. The effect of TNP-ATP was most pronounced for the 10 g force, while the effect of 5-BDBD reached significance for both the 10 g and 15 g forces. In addition, PWF was significantly lower in TNP-ATP treated rats at the 1 hour and 3 hour time points for the 10 g force. 5-BDBD also significantly lowered PWF compared to ET-1 alone, an effect that was seen at the earlier time points of 30 min and 1 hour. The difference in timing for the effects of TNP-ATP and 5-BDBD may reflect pharmacokinetic differences, but also may result from the difference in administration. TNP-ATP was pre- and co-injected, while 5-BDBD could only be pre-injected due to differences in solubility compared to ET-1. Antagonist effects were most pronounced for the 10 g force. The lower and higher PWF seen for 4 g and 15 g forces may have made it more difficult to see a significant effect of these antagonists on ET-1-induced increases in PWF. Both TNP-ATP and 5-BDBD failed to reduce mechanical sensitivity when administered in the absence of ET-1. These data imply that endogenous release of ATP acting through P2X4 receptors in the skin contributes to the mechanical sensitization caused by ET-1 injection.

We have previously shown that subcutaneous injection of CGRP and glutamate receptor antagonists also reduce the mechanical hypersensitivity caused by ET-1 injection in the rat hind paw [18]. In addition, ET-1 was found to increase the release of CGRP and glutamate from isolated DRG neurons in culture, suggesting that ET-1 injection may cause an increase in the levels of these signaling molecules in the skin. In the current study, we show that exposure of ND7/104 and mDRG neurons to ET-1 increases their response to ATP. Thus, ET-1 appears to cause mechanical hypersensitivity both by increasing the release of certain endogenous algogenic molecules in the skin (in the case of CGRP and glutamate), as well as by increasing the sensitivity of nociceptive nerve endings to some of these molecules (in the case of ATP), in addition to ET-1’s effects on neuronal ion channels [10; 35].

Our results support the hypothesis that endogenous release of ATP in the skin contributes to ET-1-induced mechanical hypersensitivity. Cutaneous ATP has also been implicated in mechanical hypersensitivity that occurs in other elevated pain states that involve sensitization of nociceptive nerve endings, including inflammation and nerve injury [4; 6; 14; 26]. Our data suggest that endogenous ATP release could also contribute to pain under conditions of elevated ET-1, such as post-incision and inflammatory pain [25].

The source(s) of ATP release in the skin that contributes to ET-1-induced mechanical hypersensitivity remains unidentified. The terminals of nociceptive fibers are one potential source, but mechanically stimulated release from keratinocytes is another intriguing possibility. Keratinocytes surround nociceptive nerve terminals in the epidermis and have been demonstrated to release ATP upon mechanical stimulation, making them prime candidates [20].

Conclusion

This study shows that ET-1 sensitizes neuronal responses to the algogenic molecule ATP and provides evidence that this mechanism contributes to ET-1-induced mechanical hypersensitivity in the rat hind paw. Sensitization to ATP by ET-1 is mediated through ET_A receptors, but is independent of the ET-1-evoked rise in intracellular calcium. The sources of cutaneous ATP that underlie mechanical hypersensitivity induced by ET-1, as well as other pain models involving peripheral sensitization, remain undetermined and represent new potential therapeutic targets for future research. ET-1 may also sensitize ATP responses in other cell types that co-express ET_A and purinergic receptors.