Livne A A, Aharonovitz O
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Biochim Biophys Acta. 1992 Apr 30;1135(1):13-8. doi: 10.1016/0167-4889(92)90160-d.
The Na+/H+ exchange time-course of BCECF-loaded human platelets, suspended in isotonic media containing NaCl and sodium propionate and activated by intracellular acidification, was measured spectrofluorimetrically. Sequential alkalinization rates decline exponentially as a function of the changing intracellular pH (pHi) and its linear expression (log rate vs. pHi) extrapolates reproducibly to the pHi set point for the Na+/H+ exchange activation. The set point of control platelets (7.28 +/- 0.01) is shifted rapidly (discernibly less than or equal to 30 s) and markedly to alkaline pHi (7.62 +/- 0.03) by PMA, that activates protein kinase C and is shifted to acidic pHi (7.05 +/- 0.01) by staurosporine, which inhibits protein kinases. The addition of 5-N-(3-aminophenyl)amiloride reveals that the alkalinization measured is predominantly Na+/H+ exchange with only a minute contribution (delta pHi = 0.012 +/- 0.002 in 1 min) of an acid loading component, at pHi greater than 7.2. The results support recent studies concluding that the set point indeed reflects the phosphorylation state of the Na+/H+ exchanger.
采用荧光分光光度法测量了悬浮于含氯化钠和丙酸钠的等渗介质中、经细胞内酸化激活的 BCECF 负载人血小板的 Na⁺/H⁺交换时程。随着细胞内 pH（pHi）的变化，连续碱化速率呈指数下降，其线性表达式（log 速率与 pHi）可重复外推至 Na⁺/H⁺交换激活的 pHi 设定点。对照血小板的设定点（7.28±0.01）被激活蛋白激酶 C 的佛波酯迅速（明显小于或等于 30 秒）且显著地移至碱性 pHi（7.62±0.03），而被抑制蛋白激酶的星形孢菌素移至酸性 pHi（7.05±0.01）。添加 5 - N -（3 - 氨基苯基）氨氯吡咪显示，在 pHi 大于 7.2 时，所测得的碱化主要是 Na⁺/H⁺交换，酸负载成分的贡献极小（1 分钟内δpHi = 0.012±0.002）。这些结果支持了最近的研究结论，即设定点确实反映了 Na⁺/H⁺交换体的磷酸化状态。
