Sphingosine 1-phosphate receptor 2 antagonist JTE-013 increases the excitability of sensory neurons independently of the receptor

Abstract

Previously we demonstrated that sphingosine 1-phosphate receptor 1 (S1PR(1)) played a prominent, but not exclusive, role in enhancing the excitability of small-diameter sensory neurons, suggesting that other S1PRs can modulate neuronal excitability. To examine the potential role of S1PR(2) in regulating neuronal excitability we used the established selective antagonist of S1PR(2), JTE-013. Here we report that exposure to JTE-013 alone produced a significant increase in excitability in a time- and concentration-dependent manner in 70-80% of recorded neurons. Internal perfusion of sensory neurons with guanosine 5'-O-(2-thiodiphosphate) (GDP-β-S) via the recording pipette inhibited the sensitization produced by JTE-013 as well as prostaglandin E(2). Pretreatment with pertussis toxin or the selective S1PR(1) antagonist W146 blocked the sensitization produced by JTE-013. These results indicate that JTE-013 might act as an agonist at other G protein-coupled receptors. In neurons that were sensitized by JTE-013, single-cell RT-PCR studies demonstrated that these neurons did not express the mRNA for S1PR(2). In behavioral studies, injection of JTE-013 into the rat's hindpaw produced a significant increase in the mechanical sensitivity in the ipsilateral, but not contralateral, paw. Injection of JTE-013 did not affect the withdrawal latency to thermal stimulation. Thus JTE-013 augments neuronal excitability independently of S1PR(2) by unknown mechanisms that may involve activation of other G protein-coupled receptors such as S1PR(1). Clearly, further studies are warranted to establish the causal nature of this increased sensitivity, and future studies of neuronal function using JTE-013 should be interpreted with caution.

Fig. 1.

JTE-013 enhances the excitability of capsaicin-sensitive small-diameter sensory neurons. A: a representative recording in which the ramp of depolarizing current evoked 3 action potentials (APs) under control conditions, whereas after a 10-min exposure to 100 nM JTE-013 the number of APs increased to 10 (right). B summarizes the sensitizing actions of JTE-013 over a 15-min recording period. There was no significant difference between the number of APs at the 2, 5, 10, and 15 min time points. The number of neurons at each time point are as follows: control 11, 2 min 6, 5 min 11, 10 min 11, and 15 min 9. C: the number of evoked APs after exposure to JTE-013 normalized to their respective control values; these are the same neurons as shown in B. Note that there were no recordings obtained at 2 min for JTE-013-insensitive neurons. *Significant difference compared with the control condition (P < 0.001, ANOVA on ranks).

Fig. 2.

JTE-013 augments excitability in a time- and concentration-dependent manner. A: number of evoked APs for the different times and concentrations of JTE-013. The data shown represent only the mean values; the SE is not shown for clarity of presentation. The number of neurons comprising the results for each concentration are as follows: vehicle 5, 1 nM 4, 3 nM 5, 10 nM 10, 100 nM 11, 1,000 nM 8. B: number of evoked APs obtained for the different times and concentrations of JTE-013 normalized to their respective control values. The data were obtained from the neurons shown in A and represent means ± SE. There was no difference in either the number of evoked APs or the normalized number of evoked APs over the recording periods for the vehicle (P = 0.20 and P = 0.24 for the number and the normalized number, respectively, ANOVA), for 1 nM JTE-013 (P = 0.38 and P = 0.27 for the number and the normalized number, respectively, ANOVA), and for 3 nM JTE-013 (P = 0.16 for both for the number and the normalized number, ANOVA on ranks). There was a significant difference in both the number and normalized number of APs for treatment times at 5, 10, and 15 min for 10, 100, and 1,000 nM JTE-013 compared with their control values (P < 0.001 ANOVA on ranks, Dunn's all pairwise test). C summarizes the increase in the normalized number of APs as a function of JTE-013 concentration for treatment times of 10 and 15 min.

Fig. 3.

Sphingosine 1-phosphate (S1P) does not cause a further increase in AP firing after treatment with JTE-013. In a separate series of experiments, 7 sensory neurons were exposed to 100 nM JTE-013 over a 15-min recording period. After the recoding at 15 min, these neurons were exposed to 1 μM S1P and recordings were obtained over the next 10 min. The data represent means ± SE. *Significant difference from the control values [P < 0.001, repeated-measures (RM) ANOVA].

Fig. 4.

Internal perfusion with guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S) blocks the increased excitability produced by JTE-013. A demonstrates that internal perfusion with 3 mM GDP-β-S prevents the increase in excitability produced by 1 μM PGE₂, which is known to act via the Gs-cAMP-PKA pathway. For the normal control condition, n = 5; for internal perfusion with GDP-β-S, n = 11. B shows that internal perfusion with GDP-β-S blocks the increase in AP firing produced by 100 nM JTE-013. The numbers of neurons at each time point are as follows: control through 12 min 8 neurons, 15 min 7, and 20 min 6. *Significant difference compared with the control condition (P < 0.05, RM ANOVA).

Fig. 5.

Pertussis toxin (PTX) and the S1P receptor 1 (S1PR₁) antagonist W146 block the JTE-013-induced increase in excitability. A: 1 group of sensory neurons were treated with 200 ng/ml PTX for 24 h, while the untreated neurons were composed of 2 groups: 1 group was obtained from neurons isolated from the same tissue harvests as those for the PTX neurons, and another group was derived from the concentration-response experiments for 10 nM JTE-013. These 2 groups were not significantly different for all time points (P > 0.05, ANOVA) and were combined. The numbers of neurons in the untreated group were as follows: control 12, JTE-013 12, 12, and 11 at 2, 6, and 10 min, respectively, and S1P 7. The numbers of neurons in the PTX-treated group were as follows: control 12, JTE-013 12, and S1P 6. *Significant difference between the control and after exposure to JTE-013 within the untreated group (P < 0.001, ANOVA Holm-Sidak all pairwise). To compare the untreated and PTX-treated neurons, an ANOVA was used. There was a significant difference (P < 0.001, Holm-Sidak all pairwise test) between the untreated neurons at 6 and 10 min from all PTX-treated time points. Untreated neurons at 2 min were significantly different from the PTX-treated controls but not PTX-treated neurons at 2, 6, and 10 min. The untreated and PTX-treated controls were not different. B: pretreatment with the selective S1PR₁ antagonist W146, but not its inactive analog W140, blocks the effects of JTE-013. A within-group statistical analysis indicated that in the presence of 1 μM W140 JTE-013 significantly increased AP firing for the recordings obtained at 2, 5, 10, and 15 min compared with the control (P < 0.001, ANOVA Holm-Sidak all pairwise test), whereas there was no difference between the number of APs recorded at 2, 5, 10, and 15 min. The numbers of neurons in the W140-treated group were as follows: control 10, JTE-013 2 min 10, 5 min 10, 10 min 7, and 15 min 5. In contrast, a within-group analysis showed that JTE-013 had no effect on the number of evoked APs in the presence of 1 μM W146 (P = 0.66 ANOVA). The numbers of neurons in the W146-treated group were as follows: control 13, JTE-013 2 min 13, 5 min 13, 10 min 12, and 15 min 12. A comparison between the W140 and W146 groups indicated that in the presence of W140 JTE-013 significantly increased the number of evoked APs at 5, 10, and 15 min compared with all time points for W146 treatment (P < 0.001, ANOVA Holm-Sidak all pairwise test). For the W140 2 min recordings, the difference was significant for the W146 control, 2, 10, and 15 min but not 5 min time points. There was no difference between the W140 and W146 control groups.

Fig. 6.

Single-cell RT-PCR demonstrates that neurons sensitized by JTE-013 do not express the mRNA for S1PR₂. A: current-clamp recordings obtained from 2 representative neurons, cell 1 (C1) and cell 2 (C2), in which a 10-min exposure to 100 nM JTE-013 increased the number of APs evoked by the current ramp. B: the RT-PCR results obtained for these 2 individual neurons. Neither C1 nor C2 expressed the mRNA for S1PR₂ (top), but they did express HPRT (bottom). As positive controls S1PR₂ and HPRT were detected in mRNA isolated from intact dorsal root ganglia (DRG) and liver. Lanes labeled B (blank) represent reactions performed in the absence of any template.

Fig. 7.

JTE-013 increases the sensitivity to mechanical but not thermal stimulation of the hindpaw. A: results obtained from 5 rats undergoing mechanical stimulation of the hindpaw with von Frey filaments. The time of JTE-013 injection is represented as the dashed line at time 0. B: results obtained for the thermal testing before and after injection of JTE-013. *Significant difference compared with the untreated control conditions at 4 and 2 days prior to injection (P < 0.05, 2-way RM ANOVA).

Fig. 8.

S1P-induced inhibition of B16 cell migration is blocked by JTE-013 in the wound healing assay. A and B: 2 representative results (from a total of 7 assays). Panels labeled Control show the untreated controls at 0 and 48 h after the wound. Panels labeled S1P show the inhibition of migration produced by 100 nM S1P 48 h after the wound. Panels labeled S1P + JTE-013 demonstrate that 100 nM JTE-013 blocks the inhibition of cell migration produced by S1P after 48 h. Panels labeled JTE-013 show that cell migration after 48 h is not affected by exposure to 100 nM JTE-013 alone.