Grinstein S, Goetz-Smith J D, Stewart D, Beresford B J, Mellors A
J Biol Chem. 1986 Jun 15;261(17):8009-16.
In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport.
在淋巴细胞中，Na⁺/H⁺逆向转运体可被12 - O - 十四烷酰佛波醇13 - 乙酸酯（TPA）和渗透性收缩所刺激。由于TPA通过刺激蛋白激酶C起作用，我们进行了实验以确定蛋白磷酸化是否也是逆向转运体渗透性刺激的基础。我们发现，在用TPA处理的细胞中标记的膜多肽中，至少有一个也会因高渗收缩而发生磷酸化。在这两种情况下，磷酸化都是碱不稳定的，并且与丝氨酸和苏氨酸残基相关。我们测试了收缩激活磷脂酶C从而通过释放二酰基甘油刺激蛋白激酶C的可能性。在高渗处理的细胞中未检测到磷脂酰肌醇4,5 - 二磷酸水平的降低。此外，在收缩的细胞中，包括肌醇三磷酸在内的肌醇磷酸浓度没有改变。因此，收缩似乎不会激活磷脂酶C。虽然TPA诱导可溶性蛋白激酶C在细胞内重新分布，但在渗透性激活的细胞中未检测到这种效应。得出的结论是，Na⁺/H⁺逆向转运体的渗透性刺激与一种激酶激活蛋白磷酸化有关，该激酶与蛋白激酶C相似但不相同。在无Na⁺或含氨氯吡脒的培养基中进行的实验表明，磷酸化不是逆向转运体激活的结果。
