Extracellular signal-regulated kinases mediate the enhancing effects of inflammatory mediators on resurgent currents in dorsal root ganglion neurons

Abstract

Previously we reported that a group of inflammatory mediators significantly enhanced resurgent currents in dorsal root ganglion neurons. To understand the underlying intracellular signaling mechanism, we investigated the effects of inhibition of extracellular signal-regulated kinases and protein kinase C on the enhancing effects of inflammatory mediators on resurgent currents in rat dorsal root ganglion neurons. We found that the extracellular signal-regulated kinases inhibitor U0126 completely prevented the enhancing effects of the inflammatory mediators on both Tetrodotoxin-sensitive and Tetrodotoxin-resistant resurgent currents in both small and medium dorsal root ganglion neurons. U0126 substantially reduced repetitive firing in small dorsal root ganglion neurons exposed to inflammatory mediators, consistent with prevention of resurgent current amplitude increases. The protein kinase C inhibitor Bisindolylmaleimide I also showed attenuating effects on resurgent currents, although to a lesser extent compared to extracellular signal-regulated kinases inhibition. These results indicate a critical role of extracellular signal-regulated kinases signaling in modulating resurgent currents and membrane excitability in dorsal root ganglion neurons treated with inflammatory mediators. It is also suggested that targeting extracellular signal-regulated kinases-resurgent currents might be a useful strategy to reduce inflammatory pain.

Keywords: Resurgent currents; Tetrodotoxin-resistant; Tetrodotoxin-sensitive; dorsal root ganglion; extracellular signal-regulated kinases; inflammatory mediators; protein kinase C.

Figure 1.

Expression pattern of resurgent currents in DRG neurons. Resurgent and regular sodium current were recorded from small (diameter: 20 to 30 µm) and medium (diameter: 35 to 45 µm) DRG neurons dissociated from adult rats. In all the small neurons, both fast (TTX-S) and slow (TTX-R) regular sodium currents were expressed (a, inset). However, only a slow (TTX-R) resurgent current was recorded from small DRG neurons. In the medium DRG neurons that expressed only fast (TTX-S) regular sodium currents ((b), inset), a fast (TTX-S) resurgent current was recorded (b). In the medium DRG neurons that expressed both fast (TTX-S) and slow (TTX-R) regular sodium currents ((c) and (d) insets), two types of expression pattern of resurgent currents were recorded. In the majority (>80%) of such neurons, a single slow (TTX-R) resurgent current was recorded (c). In other cells (<20%), both a fast (TTX-S) and a slow (TTX-R) components of resurgent currents were recorded (d). The voltage protocol for recording resurgent currents (Bottom, left) includes a series of repolarization voltage steps (+15 to −70 mV decreased by 5-mV steps, 450 ms) from a depolarization step (−100 mV to +30 mV, 20 ms). The regular sodium currents were recorded using a standard steady-state inactivation protocol that depolarized neurons to 0 mV after 500 ms pre-holding at −130 to −5 mV with an increment of 5 mV (Bottom, right). Scales for the regular sodium currents: horizontal scale, 5 ms; vertical scale, 20 nA.

Figure 2.

Effects of ERK inhibition on TTX-S resurgent currents in DRG neurons. Representative TTX-S, fast resurgent currents were recorded from medium DRG neurons ((a) to (d)). The resurgent currents were elicited by a series of repolarization voltage steps (+15 to −70 mV decreased by 5-mV steps, 450 ms) from a depolarization step (−100mV to +30 mV, 20 ms). ERK inhibitor U0126 (10 µM) and its inactive control U0124 (10 µM) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In the presence of U0124, the TTX-S resurgent currents were enhanced by a group of inflammatory mediators (IMs) including 1 µM bradykinin, 10 µM 5-HT, 10 µM histamine, 10 µM PGE2, and 5 µM ATP. (c and d) U0126 completely prevented the enhancing effects of inflammatory mediators on the TTX-S resurgent currents. Note that U0126 also significantly reduced TTX-S resurgent currents compared to U0124. (e) Statistical data of ratio resurgent currents were compared between groups with or without inflammatory mediators. Ratio resurgent currents were defined as peak resurgent currents normalized to peak TTX-S transient currents (maximal steady-state inactivation currents) in the same cells (the same below). The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, *P < 0.05 (vs. U0124).

Figure 3.

Effects of PKC inhibition on TTX-S resurgent currents in DRG neurons. Representative TTX-S, fast resurgent currents were recorded from medium DRG neurons (a–d). The resurgent currents were elicited by the same voltage protocol as described in Figure 1. PKC inhibitor BIM I (1 µM) and its control DMSO (1:1000) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In DMSO control condition, the TTX-S resurgent currents were enhanced by inflammatory mediators (IMs). (c and d) BIM I seemed to partially prevent the enhancing effects of inflammatory mediators on the TTX-S resurgent currents. (e) The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, *P < 0.05 (vs. DMSO). DMSO: dimethyl sulfoxide; BIM I: bisindolylmaleimide I.

Figure 4.

Effects of ERK inhibition on TTX-R resurgent currents in medium DRG neurons. Representative TTX-R, slow resurgent currents were recorded from medium DRG neurons (a–d). The resurgent currents were elicited by the same voltage protocol as described in Figure 1. ERK inhibitor U0126 (10 µM) and its inactive control U0124 (10 µM) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In the presence of U0124, the TTX-R resurgent currents were enhanced by inflammatory mediators (IMs). (c and d) U0126 completely prevented the enhancing effects of inflammatory mediators on the TTX-R resurgent currents. Note that U0126 also reduced TTX-R resurgent currents compared to U0124 insignificantly (P < 0.1). (e) The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, *P < 0.05 (vs. U0124).

Figure 5.

Effects of PKC inhibition on TTX-R resurgent currents in medium DRG neurons. Representative TTX-R, slow resurgent currents were recorded from medium DRG neurons (a–d). The resurgent currents were elicited by the same voltage protocol as described in Figure 1. PKC inhibitor BIM I (1 µM) and control DMSO (1:1000) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In DMSO control condition, the TTX-R resurgent currents were enhanced by inflammatory mediators (IMs). (c and d) BIM I seemed to partially prevent the enhancing effects of inflammatory mediators on the TTX-R resurgent currents. (e) The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, *P < 0.05 (vs. DMSO). DMSO: dimethyl sulfoxide; BIM I: bisindolylmaleimide I.

Figure 6.

Effects of ERK inhibition on TTX-R resurgent currents in small DRG neurons. Representative TTX-R, slow resurgent currents were recorded from small DRG neurons (a–d). The resurgent currents were elicited by the same voltage protocol as described in Figure 1. DRG neurons-exhibited slow resurgent currents (TTX-R) were chosen to study. ERK inhibitor U0126 (10 µM) and its inactive control U0124 (10 µM) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In the presence of U0124, the TTX-R resurgent currents were enhanced by inflammatory mediators (IMs). (c and d) U0126 completely prevented the enhancing effects of inflammatory mediators on the TTX-R resurgent currents. (e) The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, *P < 0.05 (vs. U0124).

Figure 7.

Effects of PKC inhibition on TTX-R resurgent currents in small DRG neurons. Representative TTX-R, slow resurgent currents were recorded from small DRG neurons (a–d). The resurgent currents were elicited by the same voltage protocol as described in Figure 1. PKC inhibitor BIM I (1 µM) and control DMSO (1:1000) were pretreated for 15 min in culture medium and were maintained in the recording chamber. (a and b) In DMSO control condition, the TTX-R resurgent currents were enhanced by inflammatory mediators (IMs). (c and d) BIM I completely prevented the enhancing effects of inflammatory mediators on the TTX-R resurgent currents. (e) The data were presented as mean ± standard error of the mean. Student’s t-test was used to compare the difference, **P < 0.01 (vs. DMSO). DMSO: dimethyl sulfoxide; BIM I: bisindolylmaleimide I.

Figure 8.

Effects of ERK inhibition on repetitive firing of small DRG neurons in the presence of inflammatory mediators. Small DRG neurons were recorded under whole-cell current clamp conditions. A three-time rheobase current was injected and the representative responses from neurons in the presence of U0124 and U0126 were shown (a and b). U0126 significantly reduced the number of action potentials (APs) (a–c) without changing rest membrane potential (RMP) (d) or rheobase (e), significantly. The data were presented as mean ± standard error of the mean. The data were from 20 neurons and 5 cell cultures for each group. Student’s t-test was used to compare the difference, *P < 0.05.