Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
J Cell Physiol. 2013 Feb;228(2):428-38. doi: 10.1002/jcp.24147.
Preimplantation mouse embryos are particularly sensitive to increased osmolarity within their normal physiological range. The detrimental effects can be alleviated by organic osmolytes such as glycine transported into early embryos, an effect thought to be due to the organic osmolyte replacing a portion of intracellular inorganic ions accumulated during acute cell volume regulation. However, no mechanism of cell volume regulation dependent on inorganic ions has been identified in preimplantation embryos. We found that decreased cell volume rapidly activated Na(+)/H(+) exchange in preimplantation mouse embryos. This activity was likely mediated by the NHE1 (Slc9a1) isoform, since it was blocked by the highly selective NHE1 inhibitor, cariporide, which also inhibited the ability of the 1-cell embryo to maintain cell volume. How NHE1 is activated by decreased cell volume is not generally well understood. Full activation of NHE1 by decreased cell volume in 2-cell mouse embryos required the activity of the tyrosine kinase Janus kinase 2 (Jak2), since NHE1 activation was inhibited by the general tyrosine kinase inhibitor genistein, several selective inhibitors of Jak2, and dominant negative Jak2 expressed in 2-cell embryos. Decreased cell volume furthermore resulted in increased tyrosine phosphorylation of proteins in 2-cell embryos detected both by anti-phosphotyrosine antibody and an antibody directed against active phospho-Jak2. Thus, Jak2 apparently serves as a cell volume sensor in embryos. Evidence from pharmacological inhibitors further indicated that NHE1 activation by decreased cell volume was dependent on calmodulin activity, likely downstream of Jak2, and required active phospholipase C.
胚胎植入前的小鼠胚胎对其正常生理范围内渗透压的增加特别敏感。这种有害影响可以通过有机渗透物来缓解，例如甘氨酸被转运到早期胚胎中，这种效应被认为是由于有机渗透物取代了在急性细胞体积调节过程中积累的部分细胞内无机离子。然而，在胚胎植入前阶段，尚未发现依赖于无机离子的细胞体积调节机制。我们发现，细胞体积的减小会迅速激活胚胎植入前的小鼠胚胎中的 Na(+)/H(+)交换。这种活性可能是由 NHE1（Slc9a1）同工型介导的，因为它被高度选择性的 NHE1 抑制剂 cariporide 阻断，cariporide 还抑制了 1 细胞胚胎维持细胞体积的能力。目前尚不清楚细胞体积减小如何激活 NHE1。在 2 细胞小鼠胚胎中，通过细胞体积减小使 NHE1 完全激活需要酪氨酸激酶 Janus 激酶 2（Jak2）的活性，因为 NHE1 的激活被广谱酪氨酸激酶抑制剂 genistein、几种 Jak2 的选择性抑制剂和在 2 细胞胚胎中表达的显性失活 Jak2 抑制。此外，细胞体积减小导致 2 细胞胚胎中蛋白质的酪氨酸磷酸化增加，通过抗磷酸酪氨酸抗体和针对活性磷酸化 Jak2 的抗体均可检测到。因此，Jak2 显然在胚胎中充当细胞体积传感器。来自药理学抑制剂的证据进一步表明，通过细胞体积减小激活 NHE1 依赖于钙调蛋白活性，可能是 Jak2 的下游，并且需要活性磷脂酶 C。
