Discriminating between Capacitative and Arachidonate-activated Ca<sup>2+</sup> Entry Pathways in HEK293 Cells

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca2+entry can account for the influx of Ca2+seen at low agonist concentrations, such a those typically producing [Ca2+]oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca2+entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca2+under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca2+-stimulated type VIII adenylyl cyclase for Ca2+entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) 271, 12438–12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca2+]or to the release of Ca2+from internal stores but was specifically dependent on the induced capacitative entry of Ca2+. In marked contrast, the arachidonate-induced entry of Ca2+completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca2+occurs via an entirely distinct influx pathway.

我们最近质疑受体激活的Ca2+内流的电容性或储存调控模型是否能够解释在低激动剂浓度下（例如那些通常产生[Ca2+]振荡的浓度）所观察到的Ca2+内流。相反，我们已经确定了一种花生四烯酸调控的、非电容性的Ca2+内流机制，它似乎在这些条件下专门负责受体激活的Ca2+内流。然而，不清楚这两个系统是反映了不同的内流途径的活性，还是仅仅是调节一个共同途径的不同机制。因此，我们利用Ca2+刺激的VIII型腺苷酸环化酶对通过电容性途径发生的Ca2+内流的已知选择性（Fagan, K. A., Mahey, R., 和Cooper, D. M. F. (1996) 271, 12438–12444）来试图区分HEK293细胞中的这两种内流机制。与早期报道一致，我们发现毒胡萝卜素诱导腺苷酸环化酶活性大约增加3倍，这与[Ca2+]的整体变化或内部储存中Ca2+的释放无关，而是特别依赖于诱导的电容性Ca2+内流。形成鲜明对比的是，花生四烯酸诱导的Ca2+内流尽管产生的内流速率比毒胡萝卜素诱导的要高得多，但完全不影响腺苷酸环化酶活性。这些数据表明花生四烯酸激活的Ca2+内流是通过一个完全不同的内流途径发生的。