Wiley J S, Gargett C E, Zhang W, Snook M B, Jamieson G P
Sydney University Department of Medicine, Nepean Hospital, Penrith, New South Wales 2750, Australia.
Am J Physiol. 1998 Nov;275(5):C1224-31. doi: 10.1152/ajpcell.1998.275.5.C1224.
Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and 133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine 5'-O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 microM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 microM) at 37 degreesC, and the delay was longer at 24 degreesC. ATP addition 2-10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10-20 microM). P2Z/P2X7 inhibitors, KN-62 and 5-(N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.
已知细胞外ATP通过P2Z受体触发胸腺细胞和淋巴细胞凋亡，在该受体上ATP是部分激动剂，其最大反应仅为完全激动剂3'-O-(4-苯甲酰基)苯甲酰腺苷5'-三磷酸（BzATP）的70%。这种溶细胞受体及其相关离子通道对Ca2+（和Ba2+）具有选择性，但也能通透大小达溴化乙锭阳离子（314 Da）的分子。逆转录聚合酶链反应显示淋巴细胞P2Z与人单核细胞最近克隆的hP2X7基因相同。当人白血病B淋巴细胞与ATP和133Ba2+一起孵育时，立即出现同位素流入。当在加入同位素前10分钟加入ATP时，流入量增加了45%。时间分辨流式细胞术用于检测与BzATP或部分激动剂ATP、2-甲硫基腺苷5'-三磷酸或腺苷5'-O-(3-硫代三磷酸)一起孵育的细胞中溴化乙锭摄取的动力学。最大有效浓度的BzATP（50 microM）以与时间呈线性的速率诱导溴化乙锭立即摄取。相比之下，在37℃加入最大有效ATP浓度（500 microM）后，观察到溴化乙锭摄取开始前有延迟（30秒），在24℃时延迟更长。在加入溴化乙锭前2 - 10分钟加入ATP可消除延迟。其他部分激动剂的延迟更长，且与激动剂产生的最大通量呈负相关。对于次最大BzATP浓度（10 - 20 microM）也观察到延迟。P2Z/P2X7抑制剂KN-62和5-(N,N-六亚甲基)-amiloride降低了激动剂诱导的溴化乙锭摄取速率并延长了延迟。结果支持这样一种模型，即P2Z/P2X7受体的激动剂介导立即的通道开放，允许小的无机阳离子通过，随后是缓慢的进一步通透性增加，允许像溴化乙锭这样的较大通透阳离子通过。第二步的速率取决于时间和温度以及激动剂的效力和浓度，并且被拮抗剂减慢，这表明它取决于处于初始开放状态的P2Z/P2X7通道的比例。
