MacGlashan D, Lavens-Phillips S
Johns Hopkins University, Asthma & Allergy Center, Baltimore, Maryland 21224, USA.
J Leukoc Biol. 2001 Feb;69(2):224-32.
These studies examine characteristics of the quiescent period (timelag) of the free cytosolic calcium ([Ca++]i) elevation that follows stimulation of human basophils through the IgE receptor. Previous studies established that the [Ca++]i timelag was sensitive to the rate of ligand binding, but little else is known about this response characteristic. The [Ca++]i timelag could be lengthened using antigenic stimulation that is rapid but only weakly induces secretion: tenfold differences in the "strength" of the stimulus, as assessed by histamine release, are associated with threefold differences in the timelag. Inhibiting p53/56lyn kinase with low concentrations of the specific inhibitor, PP1, lengthened the [Ca++]i timelag dramatically. PP1 was also found to delay the onset of syk phosphorylation and histamine release. Staurosporine and genistein, which are known to inhibit early tyrosine kinases, had, at best, only modest effects on the [Ca++]i timelag. Specific inhibitors of protein kinase C (PKC) had no effect on the [Ca++]i timelag, and direct activation of PKC with PMA had only very modest effects on the timelag. Contrary to expectations, basophils with the so-called nonreleasing phenotype demonstrated an IgE-mediated [Ca++]i response at the single-cell level. However, the length of [Ca++]i timelag in nonreleasing basophils was threefold longer than normally found in releasing basophils. Furthermore, the [Ca++]i response was significantly more asynchronous than in releasing basophils and lacking in a sustained [Ca++]i elevation. These studies indicate that the [Ca++]i timelag following stimulation through the IgE receptor is sensitive to inhibition of lyn kinase but not other agents that have been demonstrated to inhibit early tyrosine kinases previously. However, only one characteristic of the [Ca++]i response phenotype of nonreleasing basophils--the [Ca++]i timelag but not the absence of a sustained [Ca++]i elevation--could be mimicked by inhibition of lyn kinase with PP1.
这些研究考察了通过IgE受体刺激人嗜碱性粒细胞后，游离胞质钙（[Ca++]i）升高的静止期（时间延迟）的特征。先前的研究表明，[Ca++]i时间延迟对配体结合速率敏感，但关于这种反应特征的其他方面知之甚少。可以使用快速但仅微弱诱导分泌的抗原刺激来延长[Ca++]i时间延迟：通过组胺释放评估的刺激“强度”相差10倍，与时间延迟相差3倍相关。用低浓度的特异性抑制剂PP1抑制p53/56lyn激酶，可显著延长[Ca++]i时间延迟。还发现PP1可延迟syk磷酸化和组胺释放的起始。已知抑制早期酪氨酸激酶的星形孢菌素和染料木黄酮对[Ca++]i时间延迟至多只有适度的影响。蛋白激酶C（PKC）的特异性抑制剂对[Ca++]i时间延迟没有影响，用佛波酯直接激活PKC对时间延迟只有非常适度的影响。与预期相反，具有所谓非释放表型的嗜碱性粒细胞在单细胞水平上表现出IgE介导的[Ca++]i反应。然而，非释放嗜碱性粒细胞中[Ca++]i时间延迟的长度比释放嗜碱性粒细胞中通常发现的长3倍。此外，[Ca++]i反应比释放嗜碱性粒细胞中的反应明显更不同步，并且缺乏持续的[Ca++]i升高。这些研究表明，通过IgE受体刺激后的[Ca++]i时间延迟对lyn激酶的抑制敏感，但对先前已证明抑制早期酪氨酸激酶的其他试剂不敏感。然而，用PP1抑制lyn激酶只能模拟非释放嗜碱性粒细胞[Ca++]i反应表型的一个特征——[Ca++]i时间延迟，而不能模拟缺乏持续的[Ca++]i升高这一特征。
