Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats

Abstract

We recently reported that reactive oxygen species (ROS) plays an excitatory role in modulation of the exercise pressor reflex (EPR) in normal rats. In this study, we further tested two independent hypotheses: 1) ROS interacts with EPR-related ionotropic receptors such as the purinergic receptors (P(2)) and transient receptor potential vanilloid 1 receptors (TRPV1) to indirectly modulate the EPR function; 2) ROS directly affects excitability of muscle afferents by modulating the voltage-gated sodium (Na(v)) channels. To test the first hypothesis, we performed animal experiments to investigate the effect of the SOD mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) on the pressor response to hindlimb intra-arterial (IA) injection of either α,β-methylene ATP (a P(2X) agonist) or capsaicin (a TRPV1 agonist) in decerebrate rats. To test the second hypothesis, we used the patch-clamp technique to determine the effect of ROS on Na(v) channels on the soma of muscle afferents. We also performed local microinjection of a sodium channel blocker, tetrodotoxin (TTX), into ipsilateral L4/L5 dorsal root ganglia (DRGs) to investigate whether the blockade of Na(v) channels by TTX affects the EPR function. We found that Tempol did not affect the pressor response to injection of either capsaicin or α,β-methylene ATP but significantly decreased the Na(v) current in small and medium-sized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons. A membrane-permeant superoxide dismutase, polyethylene glycol (PEG)-SOD, had an effect on the Na(v) current in these neurons similar to that of Tempol. Microinjection of TTX into L4/L5 DRGs dramatically attenuated the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots. These data suggest that ROS modulates the EPR by affecting the activity of the Na(v) channels on muscle afferents.

Fig. 1.

Representative images showing how to identify muscle afferent neurons in dorsal root ganglion (DRG) during patch-clamp experiments. Top: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled muscle afferent DRG neurons with red color in fluorescent light (right) and cells in the same field under regular light (left). Bottom: isolectin B4 (IB4) was used to further separate muscle afferent DRG neurons into 2 groups: IB4-positive neurons (indicated by white arrowhead) and IB4-negative neurons (indicated by white arrow). Digitally merged images from top right and bottom left panels are displayed in bottom right panel.

Fig. 2.

Mean data showing that hindlimb intra-arterial (IA) infusion of 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) did not affect the pressor [mean arterial pressure (MAP); A] and cardioacceleration [heart rate (HR); B] responses to injection of either capsaicin [Cap, transient receptor potential vanilloid 1 (TRPV1) agonist; 0.1 or 1.0 μg/kg] or α,β-methylene ATP (ATP, P_2X agonist; 10, 40 μg/kg) in decerebrate rats. Values are means ± SE; n = 10/group. bpm, Beats per minute.

Fig. 3.

Effect of Tempol on voltage-gated sodium (Na_v) current density in muscle afferent DRG neurons from normal rats. A: representative Na_v current recording showing that Tempol (5 mM) decreased the Na_v current in a small-sized muscle afferent DRG neuron. B: current density-voltage curves showing the effect of Tempol (5 mM) on the Na_v current in small and medium-sized muscle afferent DRG neurons (n = 24: 12 for small neurons and 12 for medium-sized neurons) from normal rats. C and D: dose-dependent effect of Tempol on the Na_v current density in muscle afferent DRG neurons from normal rats [1 mM: n = 26 (12 for small neurons and 14 for medium-sized neurons); 5 mM: n = 24 (12 for small neurons and 12 for medium-sized neurons)]. Values are means ± SE. *P < 0.05 vs. Control.

Fig. 4.

Mean data showing the effect of Tempol (1 mM) on the Na_v current in different subtypes of muscle afferent DRG neurons (small vs. medium sized, n = 12 for small and 14 for medium-sized neurons; IB4-positive vs. IB4-negative, n = 11 for IB4-positive and 15 for IB4-negative neurons). Values are means ± SE. *P < 0.05 vs. Control.

Fig. 5.

Effect of polyethylene glycol (PEG)-SOD on Na_v current density in muscle afferent DRG neurons. A: representative Na_v current recording showing that PEG-SOD (50 U/ml) decreased the Na_v current in a medium-sized muscle afferent DRG neuron. B: current density-voltage curves showing the effect of PEG-SOD (50 U/ml) on the Na_v current in small and medium-sized muscle afferent DRG neurons (n = 14: 7 for small neurons and 7 for medium-sized neurons) from normal rats. C and D: mean data showing the effect of PEG-SOD (50 U/ml) on the Na_v current in different subtypes of muscle afferent DRG neurons (small vs. medium-sized, n = 7 for small and 7 for medium-sized neurons; IB4-positive vs. IB4-negative, n = 8 for IB4-positive and 6 for IB4-negative neurons). Values are means ± SE. *P < 0.05 vs. Control.

Fig. 6.

Influences of 2 redox agents (Tempol and PEG-SOD) on the superoxide level in the L4/L5 DRGs of normal rats. Values are means ± SE; n = 5. *P < 0.05 vs. vehicle. RLU, relative light units.

Fig. 7.

Effect of microinjection of tetrodotoxin (TTX) into ipsilateral L4/L5 DRGs on the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots in decerebrate rats. A: representative recordings showing the pressor [arterial blood pressure (ABP) and MAP] and cardioacceleration (HR) responses to 30-s static contraction before and after microinjection of TTX (50 μM, 200 nl/DRG) into ipsilateral L4/L5 DRGs. B and C: mean data showing the effects of microinjection of TTX (50 μM, 200 nl/DRG) into the ipsilateral L4/L5 DRGs on the pressor (MAP; B) and cardioacceleration (HR; C) responses to 30-s static contraction. Values are means ± SE; n = 6/group. *P < 0.05 vs. before.