Background
Sphingosine-1-phosphate (S1P) is a bioactive lipid which has been shown to exert important biological functions in a variety of systems such as the immune and cardiovascular systems as well as in the regulation of cancer cells [
1-
4]. S1P can function as a primary messenger to act on a family of five G-protein-coupled receptors (S1P receptors, S1PR
1–5) (reviewed by [
5,
6]). Several recent studies also demonstrate that S1P is involved in the sensation and modulation of pain (reviewed by [
7,
8]). Previous work from our laboratory demonstrated that extracellular delivery of S1P was capable of enhancing the excitability of sensory neurons in a GDP-β-S-dependent manner [
9]. Additional studies demonstrated that S1P activation of S1PRs augmented both heat- and capsaicin-activated membrane currents in mouse sensory neurons [
10]. Application of S1P increased the firing frequency of polymodal C fibers in response to a thermal stimulus in a skin-nerve preparation, suggesting that this sensitization was not a result of immune cell invasion [
10]. Similarly, injection of S1P into the rat’s hindpaw produced edema, which is a hallmark of inflammation [
11,
12] as well as significant thermal and mechanical hyperalgesia [
10,
13]. Recent single-cell quantitative real-time PCR studies from our laboratory demonstrated that small-, medium-, and large-diameter sensory neurons can express the mRNAs for all five S1PRs wherein S1PR subtype 1 (S1PR
1) was the highest expressor in greater than 50% of these isolated single neurons [
14].
To establish which S1PR mediated the enhanced excitability produced by S1P, a study using short-interfering RNA (siRNA) to selectively knockdown expression and selective agonists demonstrated that S1PR
1 plays a crucial, but not exclusive, role in mediating neuronal sensitization. Small-diameter sensory neurons treated with siRNA targeted to S1PR
1 were unresponsive to the S1PR
1 selective agonist SEW2871; however, treatment with the more global agonist, S1P, was still capable of increasing the excitability in approximately one third of the siRNA-treated neurons [
15]. Thus, these observations indicated that S1PR
1 plays a prominent role in the S1P-induced neuronal sensitization, but there must be other S1P receptors capable of mediating the S1P-induced enhancement of excitability. The studies described below show that, in addition to S1PR
1, activation of S1PR
3 can lead to the enhancement of excitability in sensory neurons.
Methods
Isolation and maintenance of sensory neurons
Sensory neurons were harvested from young adult Sprague–Dawley rats (80 to 150 g) and from young adult mice on a C57BL/6 J background (Harlan Laboratories, Indianapolis, IN, USA). Sensory neurons isolated from the mouse were only used in the examination of membrane currents activated by S1P. Briefly, male rats or mice were killed by placing them in a chamber that was then filled with CO2. Dorsal root ganglia (DRGs) were isolated and collected in a conical tube with sterilized Puck’s solution. The tube was centrifuged for 1 min at approximately 2000 × g, and the pellet was resuspended in 1 ml Puck’s solution containing 10 U of papain (Worthington, Lakewood, NJ, USA). After a 15-min incubation at 37°C, the tube was centrifuged at 2000 × g for 1 min, and the supernatant was replaced by 1 ml F-12 medium containing 1 mg collagenase IA and 2.5 mg dispase II (Roche Diagnostics, Indianapolis, IN, USA). The DRGs were resuspended and incubated at 37°C for 20 min. The suspension was centrifuged for 1 min at 2000 × g, and the supernatant was removed. The pellet was resuspended in F-12 medium supplemented with 10% heat-inactivated horse serum and 30 ng/ml nerve growth factor (NGF) (Harlan Bioproducts, Indianapolis, IN, USA) and mechanically dissociated with a fire-polished glass pipette until all visible chunks of tissue disappeared. Isolated cells were plated onto plastic coverslips previously coated with 100 μg/ml poly-d-lysine and 5 μg/ml laminin. Cells were maintained in culture at 37°C and 3% CO2 for 18 to 24 h before electrophysiological recording. All procedures have been approved by the Animal Use and Care Committee of the Indiana University School of Medicine.
Electrophysiology
Recordings were made using the whole-cell patch-clamp technique as previously described [
16]. Briefly, a coverslip with sensory neurons was placed in a recording chamber filled with normal Ringer’s solution of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl
2, 1 MgCl
2, 10 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and 10 glucose, with pH adjusted to 7.4 using NaOH. Recording pipettes were pulled from borosilicate glass tubing (Model G85165T-4, Warner Instruments, Hamden, CT, USA). Recording pipettes had resistances of 2 to 5 MΩ when filled with the following solution (in mM): 140 KCl, 5 MgCl
2, 4 ATP, 0.3 GTP, 0.25 CaCl
2, 0.5 EGTA (calculated free Ca
2+ concentration of 100 nM, MaxChelator), and 10 HEPES, at pH 7.2 adjusted with KOH. Whole-cell voltages or currents were recorded with an Axopatch 200 or Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Data were acquired and analyzed with pCLAMP 10 (Molecular Devices, Sunnyvale, CA, USA). All drugs were applied with a VC-8 bath perfusion system (Warner Instruments, Hamden, CT, USA) unless otherwise noted. In the current-clamp experiments, the neurons were held at their resting potentials (between −45 and −65 mV), and a depolarizing current ramp (1,000 ms in duration) was applied. The amplitude of the ramp was adjusted to produce between 2 and 4 action potentials (APs) under control conditions and then the same ramp was used throughout the recording period for each individual neuron. Voltages were filtered at 5 kHz and sampled at 2 kHz. In voltage-clamp recordings, neurons were held at −60 mV. Currents were filtered at 5 kHz and sampled at 500 Hz. Additionally, the voltage-clamp recordings were digitally filtered after acquisition using a low-pass 8-pole Bessel filter function (60 Hz −3 dB cutoff) in Clampfit. At the end of each recording, the neuron was exposed to 1 μM capsaicin. This neurotoxin was used to distinguish capsaicin-sensitive sensory neurons as these neurons are believed to transmit nociceptive information [
17]. However, the correlation between capsaicin sensitivity and that a neuron is a nociceptor is not absolute. Some nociceptive neurons are insensitive to capsaicin and some capsaicin-sensitive neurons are not nociceptors [
18]. Therefore, this agent was used to define a population of small-diameter sensory neurons that could serve a nociceptive function. All results presented in this report were obtained from capsaicin-sensitive neurons, unless otherwise stated. All experiments were performed at room temperature, approximately 23°C.
siRNA treatment
The gene sequences of S1PR
2 and S1PR
3 were obtained from NCBI with the accession numbers NM_017192 and XM_225216, respectively. siRNAs targeting S1PR
2 and S1PR
3 were designed by the online tool provided by the Whitehead Institute for Biomedical Research, Cambridge, MA (
http://sirna.wi.mit.edu) [
19] and synthesized by Thermo Scientific (Waltham, MA, USA). Both siRNAs were labeled with the fluorescent tag, fluorescein, with 3′-end modification. For the siRNA targeted to S1PR
2, the sense strand was 5′-CCUUCUGGUGCUAAUCGCAUU-3′, and the antisense strand was 3′-UUGGAAGACCACGAUUAGCGU-5′. For the siRNA targeted to S1PR
3, the sense strand was 5′-CAUUCUGAUGUCCGGUAGGUU-3′, and the antisense strand was 3′-UUGUAAGACUACAGGCCAUCC-5′. The siRNA targeted to S1PR
1 was the same sequence as described in [
15] and labeled with the fluorescent tag DY547. A universal Silencer Negative Control #1 siRNA (cat #4390843, Ambion, Grand Island, NY, USA) was used as the negative control.
Neurons isolated from the rat DRG were maintained in culture in F-12 medium with 30 ng/ml NGF at 37°C for 24 h. F-12 was replaced with Opti-MEM medium (Life Technologies, Grand Island, NY, USA), and the neurons were incubated at 37°C for about 5 h for lipid transfection. The transfection reagent, metafectene (Biontex-USA, San Diego, CA, USA) and siRNA complex (5 μl, 100 nM) were prepared in 2 ml Opti-MEM. Neurons were exposed to either siRNA, negative control siRNA, or metafectene alone and maintained at 37°C for 48 h. F-12 medium was used to wash out the metafectene and the siRNA; neurons were then maintained in F-12 medium with 10% heat-inactivated horse serum and 30 ng/ml NGF. Neurons were incubated for an additional 48 h before real-time quantitative PCR (qPCR) or patch-clamp experiments were performed.
cDNA generation from siRNA-treated cells
Sensory neurons that had undergone siRNA treatments were collected for real-time qPCR measurements. The F-12 medium was aspirated from the cell-culture dish, and neurons were washed with PBS solution. Total RNA from the cells was extracted by using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. The concentration of each individual RNA from different treatments was measured with a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). To eliminate contamination by genomic DNA, 500 ng of RNA was treated with 1 μl DNase I (Invitrogen, cat. #18068-015) in a 10-μl reaction at room temperature for 15 min. The reaction was terminated by adding 1 μl 25 mM ethylenedinitrilo-tetraacetic acid (EDTA), and the reaction mixture was incubated at 65°C for 10 min. To generate cDNA from RNA, the DNase-I-treated RNA template was mixed with 1 μl iScript reverse transcriptase in a 20-μl reaction (iScript cDNA Synthesis Kit cat #170-8891, Bio-Rad, Hercules, CA, USA). The reaction protocol was as follows: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min.
Pre-amplification of cDNA from siRNA-treated cells
A 0.5X pooled assay mix was prepared by adding 2 μl of 20X TaqMan® Gene Expression Assay for each gene of interest (GOI) to Tris-EDTA (TE) buffer pH 8.0, final volume 80 μl. All Gene Expression Assays are labeled with the reporter dye FAM, except for hypoxanthine phosphoribosyltransferase 1 (HPRT) which was labeled with the reporter dye VIC. To each 1 μl (25 ng) of cDNA, 5 μl of 2X Pre-amp Master Mix (Life Technologies, Grand Island, NY, USA, cat #4391128), 1 μl of 0.5X pooled assay mix, and 3 μl nuclease-free H2O (Ambion, cat #9932) were added. After a 10-min incubation at 95°C, 14 cycles of 95°C/15 s and 60°C/4 min were run, followed by storage at −20°C.
TaqMan quantitative qPCR
The pre-amplified cDNA was diluted fivefold with nuclease-free H
2O, and 2.5 μl of the dilution was used as the template in a 10-μl qPCR reaction also containing 5 μl 2X Taqman Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA, cat #4369514), 0.5 μl 20X TaqMan GOI Assay, and 2 μl nuclease-free water. A positive control template was 25 ng of pooled rat lung cDNA. Reactions were run in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The thermal-cycling condition was 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The quantification cycle (Cq) values of various GOIs were obtained at the threshold where the value of normalized fluorescence emission generated by FAM or VIC (ΔRn) reached 0.3. The expression of different genes was calculated based on the number of copies of each gene where Number of Copies = (Primer Efficiency)
−Cq. The relative expression of the GOI was determined by dividing the average copy number of the GOI by that of the reference genes, acidic ribosomal phosphoprotein P0 (Arbp) or HPRT. Efficiencies of each primer pair were determined from the slope of a seven-point standard curve (details described in [
14]).
Data analysis
Data are presented as the means ± standard error of the mean (SEM). Statistical differences in the mRNA expression levels between the control groups and the treatment groups were determined by either Student’s t-test or an analysis of variance (ANOVA). Statistical differences between the control recordings and those obtained under various treatment conditions were determined by either an ANOVA or a repeated measures (RM) ANOVA whenever appropriate. When a significant difference was obtained with an ANOVA, post hoc analyses were performed using a Holm-Sidak all-pairs test. If the data set failed the normality test, a Kruskal-Wallis one-way ANOVA on ranks was performed, followed by a Tukey or Dunn’s all pairwise test. The results were considered statistically significant when the P value was <0.05 (SigmaStat 3.5 software).
Chemicals
F-12 Nutrient Mixture (Gibco Catalog # 21700–075) was supplemented with the following per liter: 1.18 g NaHCO
3 (Sigma cat # S6014), 1X (2 mM) L-glutamine (Gibco cat # 25030–081), 50 units penicillin-50 mg/ml streptomycin (Gibco cat #15070-063), 10% heat-inactivated horse serum (Gibco cat #26050-088), 9 μg/ml 5-fluoro-2′-deoyuridine (Sigma cat # F-0503), and 21 μg/ml uridine (Sigma cat #U-3750). S1P and VPC 23019 were obtained from Avanti Polar Lipids (Alabaster, AL, USA); S1P was dissolved according to the manufacturer’s instructions (
http://www.avantilipids.com/index.php?option=com_content&view=article&id=1114&Itemid=173&catnumber=860492). Prostaglandin E
2 (PGE
2), W146, FTY720, sphingosine kinase inhibitor II (SKI-II), SEW2871, and CAY10444 were purchased from Cayman Chemical (Ann Arbor, MI, USA). CYM-5442 was purchased from Tocris Bioscience (Bristol, UK). VPC 44116 was a generous gift from Dr. Kevin R. Lynch, University of Virginia. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). PGE
2, W146, FTY720, SKI-II, SEW2871, CAY10444, VPC 23019, and VPC 44116 were dissolved in 1-methyl-2-pyrrolidinone (MPL). The MPL stock solutions were then diluted with Ringer’s solution to yield the appropriate concentrations. The vehicle, MPL was typically used at 1,000- to 5,000-fold dilutions. Our earlier studies demonstrated that MPL does not affect the potassium or sodium currents in the DRG sensory neurons [
9,
20].
Discussion
In this report, we demonstrate that S1P enhances the excitability of sensory neurons through the activation of S1PR1 and/or R3. A variety of approaches were used to isolate the contributions of specific receptors to the neuronal sensitization mediated by S1P. siRNAs targeted to individual S1PRs demonstrated that specific knockdown of the mRNA levels for S1PR1 and R3 were sufficient to prevent the sensitization produced by S1P. The results obtained with the specific agonist of S1PR3, CYM-5541, as well as pharmacological antagonists (W146, CAY10444, and the VPC compounds) are consistent with the idea that activation of S1PR1 and/or R3 augment excitability. Lastly, both FTY720 and CYM-5442 acutely increased the excitability of these sensory neurons. However, prolonged treatment with FTY720, which targets S1PR1, R3, R4, and R5, blocked the sensitization produced by either SEW2871 or S1P. In contrast, CYM-5442, which is selective for S1PR1, suppressed the effects of SEW2871 in all neurons, whereas the sensitizing actions of S1P still remained in approximately one third of the CYM-5442-treated sensory neurons. Therefore, these findings establish that the enhanced excitability produced by S1P results from the activation of S1PR1 and/or R3 but that R2, R4, and R5 are insufficient.
Our previous work indicated that S1PR
1 played a prominent, although not exclusive, role in enhancing the excitability of small-diameter sensory neurons where treatment with siRNA targeted to S1PR
1 completely blocked the SEW2871-induced sensitization, but in about one third of these siRNA treated-neurons, exposure to S1P was capable of producing significant increases in AP firing [
15]. In a real-time single-cell qPCR study of the mRNA levels of the different S1PRs in isolated sensory neurons, we found that in small- (<25 μm,
n = 18), medium- (25 to 40 μm,
n = 17), and large-diameter (>50 μm,
n = 17) neurons, S1PR
1 was the highest expressing subtype in more than half (>10) of the total individual cells in each group [
14]. In those neurons with S1PR
1 as the highest expressor, five of the ten small- and five of the ten medium-diameter neurons expressed S1PR
3 as the second highest subtype. In addition, there was a strong correlation between the expression of S1PR
1 and R
3 in both small- and medium-diameter sensory neurons (Pearson’s correlation coefficients of 0.89 and 0.92, respectively) [
14]. Thus, after S1PR
1, S1PR
3 was the second highest expressor in approximately 50% of these identified neurons. These results are consistent with our previous siRNA studies examining the functional response of S1PR
1 as well as those described above where the down-regulation of S1PR
1 by CYM-5442 yields a group of neurons that were responsive to S1P, but not SEW2871. Based on the capacities of FTY720 and CYM-5442 to act as functional antagonists, these results suggest that after CYM-5442-induced down-regulation of S1PR
1, S1PR
3 remains capable of activation. The potential differences in cellular responses mediated by S1PR
1 compared to S1PR
3 may result from coupling to different G proteins and their respective downstream effectors. S1PR
1 is believed to couple with only G
i/o whereas S1PR
3 can couple with G
i/o, G
q/11, or G
12/13, thus leading to the activation of a variety of effector systems; see reviews [
5,
63-
65]. However, the specific roles of S1PR
1 and R
3 in the regulation of neuronal excitability remain to be defined and will be the focus of future investigations.
In addition, other studies support a role for S1P-S1PR
1 in regulating the sensitivity of nociceptive sensory neurons. Opioid-induced hyperalgesia significantly decreased the latency of paw withdrawal to a thermal stimulus; this enhanced sensitivity was associated with a fourfold increase in the levels of S1P measured in the dorsal horn of the spinal cord [
66]. Both the heightened sensitivity and the increase in S1P were blocked by pretreatment with either
n-
n-dimethylsphingosine or SK-I, inhibitors of sphingosine kinases. Injection of either S1P or SEW2871 into the rat or mouse paw produced thermal hyperalgesia ([
13,
10], respectively), which was blocked by treatment with W146, a selective antagonist for S1PR
1 [
13]. Localized perfusion of the L5 DRG with S1P increased the sensitivity of the rat’s paw to mechanical stimulation (by von Frey hairs) [
67]. These authors also showed that localized injection of the inflammatory agent, zymosan, at the L5 DRG resulted in mechanical hypersensitivity of the hindpaw. However, a prior localized injection of siRNA targeted to S1PR
1 at the L4/L5 DRG significantly reduced this hypersensitivity, suggesting that S1PR
1 played a key role in the onset of this inflammatory-induced hypersensitivity [
67].
An earlier study demonstrated that both intraperitonal and intrathecal delivery of FTY720 could reduce the nociceptive behaviors associated with either the inflammatory formalin model (number of flinches) or the neuropathic spared-nerve injury model (mechanical thresholds) [
68]. Interestingly, effective doses of FTY720 did not have significant effects on the numbers of circulating white blood cells or lymphocytes, suggesting that the anti-nociceptive effects were not mediated by the immunosuppressive actions of FTY720. In contrast, the selective S1PR
1 agonist, SEW2871, had no analgesic effect on the formalin-induced hypersensitivity.
Recently, it was shown that the intrathecal injection of SEW2871 produced a hypersensitivity (both allodynia and hyperalgesia) to mechanical stimulation of the rat’s hindpaw; this hypersensitivity was blocked by the S1PR
1 selective antagonist, W146 [
69]. Interestingly, the mechanical hypersensitivity resulting from the repeated injection of the chemotherapeutic agent, paclitaxel, was also blocked by W146 in a dose-dependent manner, suggesting that S1P-S1PR
1 may play a role in the chemotherapy-induced peripheral neuropathy caused by paclitaxel. The peak of the increased sensitivity resulting from paclitaxel was associated with increased activity in the enzymes regulating the ceramide-sphingosine-S1P pathway, notably sphingosine kinase. Consistent with the idea that the paclitaxel-induced hypersensitivity was associated with S1P-S1PR
1, prior intrathecal treatment with either FTY720 or CYM-5442 blocked, in a dose-dependent manner, the increased sensitivity caused by either SEW2871 or paclitaxel. Of significance, established paclitaxel-induced hypersensitivity could be reversed by exposure to either W146, FTY720, or CYM-5442, but not SEW2871. No effect on circulating white blood cells was observed. These results in combination with those results obtained by Coste
et al. [
68] strongly support the idea that antagonism rather than activation of S1PR
1 is a key target in the suppression of this neuronal hypersensitivity. In future studies, it will be important to establish the signaling cascades that are activated by S1P-S1PR
1 and determine the specific effectors that mediate the increased sensitivity as possible therapeutic targets.
A number of studies have established that the S1P-S1PR
1 pathway plays a significant role in regulating the sensitivity of nociceptive sensory neurons through both cellular and behavioral approaches. However, in addition to our results described above, only one other study has explored the possible role of S1PR
3 in regulating the sensitivity of sensory neurons. Camprubi-Robles
et al. [
62] demonstrated that S1P, presumably through activation of S1PR
3, was capable of directly mediating a membrane current in nearly all sensory neurons isolated from the mouse DRG. Although neither the recordings of the reversal potential nor the concentration dependence were shown, application of 100 μM niflumic acid hastened the recovery phase of the S1P-induced current, suggesting that it was conducted by chloride. In current-clamp recordings, these authors report that 1 μM S1P, on average, depolarized the resting membrane potential from −54 to −36 mV with an increase in spontaneous AP firing. Our results demonstrate that FTY720 also depolarized the resting membrane potential by a similar amount (−54 to −39 mV); however, there was no enhancement of spontaneous activity, only AP firing evoked by the current ramp. We did observe a large depolarization in response to high concentrations of either CYM-5541 or SEW2871, and based on the suppressive effects of W146, this depolarization is thought to result from activation of S1PR
1. In S1PR
3−/− knockout mice, Camprubi-Robles
et al. found that S1P depolarized the neuron by only approximately 5 mV; however, no membrane current recordings from the S1PR
3−/− mice were shown. Using a fura-2-based assay, Camprubi-Robles
et al. indicate that, in normal wildtype mice, approximately 60% of the neurons were responsive to 1 μM S1P and that niflumic acid reduced this to approximately 14%. However, in the S1PR
3−/− mice, 40% of the neurons responded to S1P; if S1PR
3 specifically mediates this response, it is curious why the knockout is not more similar to the actions of niflumic acid. Although the authors claim that this S1P-mediated current was exhibited by nearly all neurons, our experiments in both rat and mouse small-diameter sensory neurons failed to detect any measurable change in membrane current after exposure to even high concentrations of S1P (10 and 100 μM). In addition, S1P failed to augment the excitability in medium- to large-diameter rat sensory neurons. The basis for this difference remains an open question. One possibility could be differences in the culture media. In the Camprubi-Robles
et al. study, sensory neurons were maintained in a synthetic serum-free medium supplemented with high levels of NGF (100 ng/ml) whereas, in our experiments, sensory neurons were maintained in an F-12 medium supplemented with 10% heat-inactivated horse serum and a lower concentration of NGF (30 ng/ml).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CL, JK, and GDN designed the study. CL, JL, and JK performed the experiments. CL, JL, JK, and GDN analyzed the data. MG provided reagents. CL, JK, and GDN wrote the manuscript. All authors read and approved the final manuscript.