Montero Mayte, Lobatón Carmen D, Gutierrez-Fernández Silvia, Moreno Alfredo, Alvarez Javier
Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Ramón y Cajal, 7, E-47005 Valladolid, Spain.
J Biol Chem. 2003 Dec 12;278(50):49972-9. doi: 10.1074/jbc.M308378200. Epub 2003 Sep 30.
In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.
在HeLa细胞中，组胺诱导肌醇1,4,5-三磷酸（InsP3）的产生，并促使内质网（ER）释放Ca2+。Ca2+释放通常呈双相性，先是快速且短暂的初始阶段，随后是更为缓慢且持久的阶段。在存在蛋白激酶C（PKC）抑制剂的情况下，包括星形孢菌素以及特异性抑制剂GF109203X和Ro-31-8220，快速阶段会持续到内质网完全排空。相反，用佛波醇12,13-二丁酸酯处理会抑制Ca2+释放。星形孢菌素对通透细胞中InsP3诱导的Ca2+释放没有影响，也不改变组胺诱导的InsP3产生。这些数据表明，组胺诱导Ca2+释放，并在短暂延迟后激活PKC以对其进行下调。一致的是，在存在PKC抑制剂的情况下，组胺诱导的Ca2+振荡幅度增加而频率降低。我们还表明，线粒体[Ca2+]对PKC调节诱导的内质网Ca2+释放变化比胞质[Ca2+]更为敏感。PKC抑制剂使组胺诱导的线粒体[Ca2+]峰值增加了4倍，但仅使胞质[Ca2+]峰值增加了20%。相反，PKC激活使线粒体[Ca2+]峰值降低了90%，而使胞质[Ca2+]峰值仅降低了50%。同样，PKC抑制剂与线粒体Ca2+单向转运体激活剂SB202190联合使用导致线粒体[Ca2+]峰值显著增加，而对胞质[Ca2+]峰值影响很小。这表明PKC抑制剂激活内质网Ca2+释放可能参与了星形孢菌素诱导的细胞凋亡。此外，这些机制允许在细胞刺激过程中对胞质和线粒体[Ca2+]进行灵活且独立的调节。
