Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca²⁺]_i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydrylreducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2′,7′-dichlorofluorescin diacetate (H₂DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca²⁺ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca²⁺ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca²⁺-sensitive fluorescence dye fura-2, the [Ca²⁺]_i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ([Cα2+]i) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the [Cα2+]i], which was inhibited by pretreatment with phenyl-N-tertbuthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced [Cα2+]i] increase was suppressed both in a calcium free solution and after depletion of the intracellular Cα2+ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red (50 μM) and capsazepine (10 μM). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.

Change in intracellular -concentration () is an essential event for egg activation and further development. ion is originated from intracellular -store via inositol 1,4,5-triphosphate receptor and/or influx via channel. This study was performed to investigate whether changes in /calmodulin dependent protein kinase II (CaM KII) activity affect influx during artificial egg activation with ethanol using monitoring system and whole-cell patch clamp technique. Under ion-omitted condition, -oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced increase was reduced. To investigate the role of CaM KII known as an integrator of - oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward current () in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that influx during fertilization might be controlled by CaM KII activity.