Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells
Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells
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DOI：
10.1038/sj.bjp.0702205
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发表时间：
1998-11-01
影响因子：
7.3
通讯作者：
Humphrey, PPA
中科院分区：
文献类型：
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作者：
Michel, AD;Chessell, IP;Humphrey, PPA
我们检测了CHO-KI细胞对P2受体激动剂2'-和3'- o -（4-苯甲酰苯甲酰）- atp （DbATP）的细胞反应，以及P2X受体mRNA的存在使用针对大鼠P2X亚基的引物进行逆转录-聚合酶链反应，检测到P2X的存在，但未检测到P2X(1)-P2X(6)亚基DbATP （EC（50类似于）100 μ M）引起非脱敏内向电流，该电流在类似于0mV的情况下反转，表明激活了非选择性阳离子通道。ATP也引起向内电流，但不如DbATP.4有效DbATP还刺激CHO-K1细胞中（45）钙（Ca-45(2+)）和DNA结合染料YO-PRO-1的积累。NaCl和MgCl2抑制了这两种反应。在280 mM蔗糖缓冲液中，45Ca（2+）的积累在加入激动剂10-20秒内可测量到，而YO-PRO-1的积累仅在8分钟后可检测到。ATP和ATP γ S也是激动剂，但效力不如DbATP，而UTP， 2-甲基硫ATP， ADP和α - β -亚甲基ATP在浓度高达100 μ m时无活性。DbATP增加CHO-K1细胞的乳酸脱氢酶释放，表明细胞裂解，尽管这种作用仅在60-90分钟后才显着6这些数据表明CHO-K1细胞表达一种内源性P2X(7)受体，该受体可被DbATP激活，引起快速内向电流和Ca-45（2+）的积累。长时间的受体激活导致对YO-PRO-1等大分子的渗透性延迟和增加，最终导致细胞裂解。重要的是，当这些细胞用于研究重组P2X受体时，应考虑内源性P2X(7)受体的存在。
1 CHO-KI cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors.2 Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X, but not P2X(1)-P2X(6) subunits.3 DbATP (EC(50 similar to)100 mu M) evoked non-desensitizing inward currents which reversed at similar to 0mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP.4 DbATP also stimulated the accumulation of (45)calcium (Ca-45(2+)) and the DNA binding dye, YO-PRO-1, in CHO-K1 cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca(2+) accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATP gamma S were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and alpha beta methylene ATP were inactive at concentrations up to 100 mu M.5 DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min.6 These data suggest that CHO-K1 cells express an endogenous P2X(7) receptor which can be activated by DbATP to cause a rapid inward current and accumulation of Ca-45(2+). Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X(7) receptor should be considered when these cells are used to study recombinant P2X receptors.