• International Journal of Oral Biology
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• 제33권3호
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• pp.117-123
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• 2008

Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In the present study, whole cell patch clamp recordings were carried out to investigate the effects of tert-buthyl hydroperoxide (t-BuOOH), an ROS, on neuronal excitability and the mechanisms underlying changes of membrane excitability. In current clamp condition, application of t-BuOOH caused a reversible membrane depolarization and firing activity in substantia gelatinosa (SG) neurons. When slices were pretreated with phenyl-N-tert-buthylnitrone (PBN) and ascorbate, ROS scavengers, t-BuOOH failed to induce membrane depolarization. However, isoascorbate did not prevent t-BuOOH-induced depolarization, suggesting that the site of ROS action is intracellular. The t-BuOOH-induced depolarization was not blocked by pretreatment with dithiothreitol (DTT), a sulfhydryl-reducing agent. The membrane-impermeant thiol oxidant 5,5-dithiobis 2-nitrobenzoic acid (DTNB) failed to induce membrane depolarization, suggesting that the changes of neuronal excitability by t-BuOOH are not caused by the modification of extrathiol group. The t-BuOOH-induced depolarization was suppressed by the phospholipase C (PLC) blocker U-73122 and inositol triphosphate ($IP_3$) receptor antagonist 2-aminoethoxydiphenylbolate (APB), and after depletion of intracellular $Ca^{2+}$ pool by thapsigargin. These data suggest that ROS generated by peripheral nerve injury can induce central sensitization in spinal cord, and t-BuOOH-induced depolarization may be regulated by intracellular $Ca^{2+}$ store mainly via $PLC-IP_3$ pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제26권1호
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• pp.25-36
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• 2022

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K⁺ currents (I_K) of human cardiac fibroblasts (HCFs), we used the wholecell mode patch-clamp technique. Application of CO delivered by carbon monoxidereleasing molecule-3 (CORM3) increased the amplitude of outward K⁺ currents, and diphenyl phosphine oxide-1 (a specific I_K blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on I_K. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3's effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced I_K activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced I_K activation. These results suggest that CO enhances I_K in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.