Korean J Physiol Pharmacol.  2017 Jul;21(4):371-376. 10.4196/kjpp.2017.21.4.371.

Reactive oxygen species increase neuronal excitability via activation of nonspecific cation channel in rat medullary dorsal horn neurons

Affiliations
  • 1Department of Dental Hygiene, Gwangyang Health Science University, Gwangyang 57764, Korea.
  • 2Department of Physiology, College of Medicine, Wonkwang University, Iksan 54538, Korea.
  • 3Department of Oral Physiology, College of Dentistry, Wonkwang University, Iksan 54538, Korea. physio1@wonkwang.ac.kr

Abstract

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion (Oâ‚‚(·âˆ’)), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO (300 µM/30 mU) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near −40 mV. X/XO-induced depolarization reduced in the presence of La³âº, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of Na⁺ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

Keyword

Nonspecific cation channel; Orofacial pain; Reactive oxygen species; Xanthine oxidase

MeSH Terms

Animals
Catalase
Facial Pain
Fluorescence
Ions
Membranes
Neurons*
Oxidoreductases
Posterior Horn Cells*
Rats*
Reactive Oxygen Species*
Sodium
Spinal Cord Dorsal Horn*
Superoxide Dismutase
Superoxides
Trigeminal Nucleus, Spinal
Xanthine Oxidase
Catalase
Ions
Oxidoreductases
Reactive Oxygen Species
Sodium
Superoxide Dismutase
Superoxides
Xanthine Oxidase

Figure

  • Fig. 1 X/XO-induced membrane depolarization is attenuated by pretreatment with ROS scavengers.The X/XO elicited a depolarization (Aa) and inward current (Ab), repeatedly. The X/XO-induced depolarization was significantly suppressed by pretreatment with PBN (Ac), SOD (Ad), and SOD+catalase (Ae). (B) Summary data obtained under the control condition of X/XO-induced depolarization and pretreatment with antioxidants. **Values are significantly different from the X/XO by independent t-test (p<0.01). *p<0.05. Means±SEM.

  • Fig. 2 X/XO-induced increase of the fluorescence intensity is attenuated by pretreatment with PBN.(A) Fluorescence intensity in the DCF-DA (green) and DHE-loaded (red) MDH cells increased on the application of X/XO (center); while PBN (right) prevented the X/XO-induced fluorescence increase (scale bars: 20 mm). (B) The results were quantitatively analyzed as percent units of DCF-DA and DHE fluorescence of the control. ***Values are significantly different from the control by paired t-test (p<0.001). ##Values are significantly different between X/XO and PBN+X/XO in DCF-DA fluorescence by paired t-test (p<0.01). ++Values are significantly different between X/XO and PBN+X/XO in DHE fluorescence (p<0.01). Means±SEM.

  • Fig. 3 The changes of neuronal excitability by X/XO are related to the chloride channel.The X/XO-induced depolarization decreased by DIDS (300 mM), an inhibitor of chloride channel within bath (Aa) or pipette solution (Ab). (B) Bar graph showing the effects of DIDS on X/XO-induced depolarization. ***Values are significantly different from the X/XO by independent t-test (p<0.001). **p<0.01. Means±SEM.

  • Fig. 4 X/XO-induced current is consistent with the opening of a nonselective cation channel.(A) X/XO-induced current from different holding potentials. At a holding potential of −40 mV, X/XO-induced current does not appeared. (B) In normal ECF solution, the current during a ramp from −120 to 40 mV increased on application of X/XO. Subtracted current (X/XO minus control) is reversed near −40 mV.

  • Fig. 5 X/XO-induced responses are involved in the influx of Na+ ions.(A) In low Na+ solution, X/XO-induced depolarization (a) and inward current (b) significantly decreased. (Ac) In La3+, a nonspecific cation channel blocker, containing solution, X/XO-induced depolarization decreased. (B) Bar graph showed that the effects of low Na+ and La3+ on X/XO-induced depolarization. ***Values are significantly different from the X/XO by independent t-test (p<0.001). **p<0.01. Means±SEM.


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