Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
J Cell Biol. 2010 Feb 22;188(4):547-63. doi: 10.1083/jcb.200908086. Epub 2010 Feb 15.
Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.
巨胞饮作用通过其对 Na(+)/H(+) 交换抑制剂的独特敏感性与其他类型的胞吞作用区分开来。然而，Na(+)/H(+) 交换与巨胞饮体形成之间的功能关系仍然不清楚。在 A431 细胞中，EGF 的刺激同时激活了巨胞饮作用和 Na(+)/H(+) 交换，升高了细胞质 pH 值并刺激了 Na(+)内流。值得注意的是，尽管 Na(+)/H(+) 交换的抑制剂阿米洛利或 HOE-694 消除了巨胞饮作用，但细胞质碱化或 Na(+)内流都不是必需的。相反，我们使用亚膜 pH 的新型探针检测到在巨胞饮作用部位积累了代谢产生的酸，Na(+)/H(+) 交换会拮抗这种作用，而当存在阿米洛利或 HOE-694 时，这种作用会被大大放大。在抑制剂存在下观察到的酸化不会改变受体结合或磷酸化，也不会显著抑制磷脂酰肌醇-3-激酶的刺激。然而，促进肌动蛋白重塑的 GTPase 的激活被发现对亚膜 pH 值非常敏感。这种敏感性使巨胞饮作用对 Na(+)/H(+) 交换抑制剂具有独特的敏感性。
