Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity.
Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity.
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氟化钠可防止受体和蛋白激酶 C 介导的人血小板 Na /H 交换器的激活，而不抑制其基本 pHi 调节活性。
DOI：
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发表时间：
1990
影响因子：
4.8
通讯作者：
J. Akkerman
中科院分区：
文献类型：
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作者：
W. Siffert;K. Jakobs;J. Akkerman
当人血小板用凝血酶或蛋白激酶C的激活剂刺激时，由于Na+/H+交换的激活，细胞溶质pH（pHi）增加。为了进一步阐明调节交换器的分子机制，我们使用氟化钠，这是一种已知的血小板中鸟嘌呤核苷酸结合蛋白（G蛋白）的激活剂。尽管NaF诱导了fura 2-负载的血小板中胞内储存位点的Ca 2+的动员，但它未能提高由2，7-双-（2-羧乙基）-5（6）-羧基荧光素负载的血小板的荧光测定的pHi。此外，当凝血酶（0.1单位/ml）或佛波酯12-O-十四酰基佛波醇13-乙酸酯（TPA）将pHi从7.13 +/- 0.05升高至7.35 +/- 0.07（n = 30）时，加入NaF（2.5-10 mM）迅速将pHi恢复至刺激前的值。相反，预孵育的血小板与低浓度的氟化钠（2.5毫米）完全防止碱化反应凝血酶或TPA。与完全阻断Na+/H+交换的乙基异丙基氨氯吡咪不同，氟化钠并不阻止pHi从人工酸负荷中恢复。因此，NaF的抑制作用仅限于受体介导的反向转运激活。为了研究NaF效应是否归因于G蛋白，将血小板与N-乙基马来酰亚胺（50 μ M）预孵育，已知N-乙基马来酰亚胺可抑制腺苷酸环化酶抑制性G蛋白。N-乙基马来酰亚胺处理不仅阻止肾上腺素对腺苷酸环化酶的抑制，而且完全逆转NaF对Na+/H+交换的抑制作用。我们的数据表明，存在一种新的G蛋白，它是由氟化物激活，并作为血小板中的Na+/H+交换的负调节器的功能。
When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pHi) increases due to activation of Na+/H+ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca2+ from intracellular storage sites in fura2-loaded platelets, it failed to raise pHi as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pHi from 7.13 +/- 0.05 to 7.35 +/- 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pHi to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na+/H+ exchange, NaF did not prevent the recovery of pHi from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na+/H+ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na+/H+ exchanger in platelets.