NG108-15細胞のATPにより誘発される非選択性陽イオン電流

Abstract

ATP at 100 μM induced a trasient [Ca2+]i increase, while at 1 mM it induced the transient increase followed by a sustained [Ca2+]i increase in NG108-15 cells. We examined the mechanism of this sustained [Ca2+] increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. Pretreatment of the cells with U73122 (PLC inhibtor) completely inhibited the trasient [Ca2+]i increase but not the sustained [Ca2+]i increase by ATP. The sustained [Ca2+]i increase was not observed in the absence of extracellular Ca2+, suggesting that this component was due to Ca2+ influx. Under the whol-cell patch clamp, 1 mM ATP induced a membrane current with the reversal potential at 12.5±0.8 mV in Tyrode solution with the Cs+ aspartate pipete solution. The ATP-induced current increased in a concentration dependent manner with EC50 value of approximately 0.75 mM ATP. When external Na+ was replaced by Li+, K+, Rb+, Cs+ or NMG+, ATP induced the current with a slight shift in the reversal potentials, suggesting that ATP activated a nonselective cation current. From the reversal potentials, the permeability ratio was calculated to be Na+ (1) > Li+ (0.93) > K+ (0.89) > Rb+ (0.56) > Cs+ (0.47) > NMG+ (0.12). Upon total replacement of external cation with Ca2+, ATP could induce the current, inicating that Ca2+ was permeable through the current. The permeability ratio of Ca2+ : Na+ was 1 : 0.6. ATP analogues which induced the current were 2MeSATP, BzATP, ATPγs and ADPβs. UTP had only a small effect and adenosine did not induce any current. α, β MetATP, β, γMetATP and ADP induced a different type of current. In fura-2 fluorescent method, ATPγs increased [Ca2+]i sustaindly but α, β MetATP and β, γ MetATP did not do so. These results indicated that ATP induced a nonselective cation current via P2Z receptor subtype. ATP-induced current was larger at lower [Mg2+]o, indicating that ATP4- was an effective agonist form of ATP. Calculated EC50 value was 21.5 μM ATP4-. We conclude that a high concentration of ATP stimulates P2Z receptor which is coupled to a class of Ca2+-permeable nonselective cation channels through which Ca2+ influx occurs in NG 10815 cells.