Bierman A J, Cragoe E J, de Laat S W, Moolenaar W H
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht.
J Biol Chem. 1988 Oct 25;263(30):15253-6.
Addition of growth factors to responsive cells in HCO3- -free media results in a rapid rise in cytoplasmic pH (pHi) caused by activation of Na+/H+ exchange. In this paper, we have examined how pHi regulation and growth factor responsiveness are affected by HCO3(-)using quiescent mouse MES-1 fibroblastic cells as a model. When cells are exposed to 25 mM HCO3-, 5% CO2, steady-state pHi reaches a new more alkaline level (by 0.25 unit) within 10 min. This rise in pHi is both Na+- and HCO3- -dependent, does not occur in Cl(-)-depleted cells, and is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by 5-(n,n-dimethyl)-amiloride, indicating the involvement of Na+-dependent HCO3-/Cl- exchange. Furthermore, the recovery of pHi from acute acid loads is accelerated by HCO3- in a Na+-dependent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive manner and is blocked in Cl(-) -depleted cells. Similar results were obtained for mouse 3T3 cells and human fibroblasts. In the presence of HCO3-/CO2 (pH 7.35), mitogens and phorbol esters fail to induce a detectable rise in pHi. However, when steady-state pHi is artificially lowered by approximately 0.4 unit, growth factors evoke significant increases in pHi due to activation of Na+/H+ exchange. In the absence of HCO3-, mitogen-induced alkalinizations are readily detectable but not when pHi is artificially elevated to the value normally observed in HCO3- media. From these results we conclude that: 1) Na+-dependent HCO3-/Cl- exchange determines steady-state pHi and acts in parallel with Na+/H+ exchange to stimulate pHi recovery from acid loading; 2) Na+-dependent HCO3-/Cl- exchange raises steady-state pHi to a level beyond the operating range of the Na+/H+ exchanger and thereby prevents growth factors from alkalinizing the cytoplasm any further. The results also imply that, unlike Na+/H+ exchange, Na+-dependent HCO3-/Cl- exchange is not activated by mitogens.
在无HCO₃⁻的培养基中，向反应性细胞添加生长因子会导致由于Na⁺/H⁺交换激活而引起细胞质pH（pHi）迅速升高。在本文中，我们以静止的小鼠MES - 1成纤维细胞为模型，研究了HCO₃⁻如何影响pHi调节和生长因子反应性。当细胞暴露于25 mM HCO₃⁻、5% CO₂时，稳态pHi在10分钟内达到一个新的更高碱性水平（升高0.25个单位）。pHi的这种升高既依赖于Na⁺也依赖于HCO₃⁻，在Cl⁻缺失的细胞中不发生，并且受到4,4'-二异硫氰基芪-2,2'-二磺酸的抑制，但不受5 -（n，n - 二甲基）-氨氯吡脒的抑制，表明涉及Na⁺依赖性HCO₃⁻/Cl⁻交换。此外，HCO₃⁻以Na⁺依赖性和对4,4'-二异硫氰基芪-2,2'-二磺酸敏感的方式加速了pHi从急性酸负荷中的恢复，并且在Cl⁻缺失的细胞中被阻断。小鼠3T3细胞和人成纤维细胞也得到了类似的结果。在HCO₃⁻/CO₂（pH 7.35）存在的情况下，有丝分裂原和佛波酯未能诱导可检测到的pHi升高。然而，当稳态pHi被人为降低约0.4个单位时，生长因子由于Na⁺/H⁺交换激活而引起pHi显著升高。在没有HCO₃⁻的情况下，有丝分裂原诱导的碱化很容易检测到，但当pHi被人为升高到在HCO₃⁻培养基中通常观察到的值时则不然。从这些结果我们得出结论：1）Na⁺依赖性HCO₃⁻/Cl⁻交换决定稳态pHi，并与Na⁺/H⁺交换并行作用以刺激pHi从酸负荷中的恢复；2）Na⁺依赖性HCO₃⁻/Cl⁻交换将稳态pHi提高到超出Na⁺/H⁺交换器工作范围的水平，从而防止生长因子进一步使细胞质碱化。结果还表明，与Na⁺/H⁺交换不同，Na⁺依赖性HCO₃⁻/Cl⁻交换不会被有丝分裂原激活。
