Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells

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.