Effects of Na+/H+ antiport and intracellular pH in the regulation of HL-60 cell apoptosis

Abstract

The roles of Na+/H+ antiport and intracellular pH in apoptosis of HL-60 cells were investigated here. We found that dimethyl amiloride, a specific Na+/H+ antiport inhibitor, induced intracellular acidification but not apoptosis; while sodium ionophore, monensin caused intracellular alkalinization as well as apoptosis in HL-60 cells. Br-A23187 and thapsigargin could induce a various degree of intracellular alkalinization through the stimulation of Na+/H+ antiport. Dimethyl amiloride blocked the intracellular alkalinization and inhibited apoptosis induced by Br-A23187 and thapsigargin. PMA also stimulated Na+/H+ antiport and induced intracellular alkalinization which was completely blocked by dimethyl amiloride and partially attenuated by PKC inhibitors. PMA could inhibit apoptosis in HL-60 cells. PMA-induced suppression of apoptosis was, however, not interfered by dimethyl amiloride, but could be abolished by PKC inhibitors. These results indicate that pHi alkalinization and/or the stimulation of Na+/H+ antiport, instead of intracellular acidification, are contributory to the induction of apoptosis. PMA-induced inhibition of apoptosis is not necessarily associated with intracellular alkalinization, but primarily due to activation of PKC. We suggest that stimulation of Na+/H+ antiport and pHi alkalinization act as facilitating factors in the induction of apoptosis.

Antileukemia activity of perillyl alcohol (POH): uncoupling apoptosis from G0/G1 arrest suggests that the primary effect of POH on Bcr/Abl-transformed cells is to induce growth arrest

“…However, care must be taken in interpreting the association between phorbol ester protection from apoptosis and PKC-mediated cellular alkalinization since TPA-induced protection from apoptosis has also been attributed to a PKCdependent mechanism other than activation of the antiport. 67,71 Nevertheless, because two different amiloride analogs inhibited the ability of TPA to protect against POHmediated cytotoxicity, we conclude that TPA protection in our model requires a functional antiport. In fact, when the antiport is inhibited, cells appear to be even more sensitive to POHmediated cytotoxicity.…”

“…70,71 In order to determine whether the POH-induced apoptosis in Bcr/Abltransformed FDC.P1 cells is accompanied by a change in cytoplasmic pH, cells treated overnight with POH and untreated controls were loaded with the pH-sensitive dye, BCECF, and intracellular pH was analyzed by flow cytometry. In both experiments, the untreated controls (Figure 4a and e) contained cell populations with a single cytoplasmic pH peak that was measured by comparing it to standards of known pH to be about 7.5.…”

“…65,[67][68][69][70][71] Thus, preventing cytoplasmic acidification is expected to protect against DNA fragmentation, but not to affect other events that precede DNA digestion, such as cell cycle arrest. 65 Our data showing (1) that apoptotic cells are totally found in the acidic subpopulation of POH treated cells, (2) that TPA can protect against both POH-mediated cell acidification and apoptosis, (3) that TPA protection from POHmediated apoptosis requires a functional Na + /H + antiport and is PKC-dependent and (4) that TPA does not affect POHmediated G1 arrest, together, are consistent with the TPA protective effect operating prior to cytoplasmic acidification and DNA fragmentation but after cell cycle arrest.…”

Chronoregulation by Asparagine Deamidation

“…These findings are notable because there is an increase in pH to as high as pH 8.0 early in several forms of apoptosis, including DNA damage-induced apoptosis (25)(26)(27)(28)(29)(30). The increase in pH is mediated by the protein Na + /H + exchanger 1 (NHE1), a ubiquitously expressed protein that regulates cytosolic pH (25)(26)(27)(28)(29). Together, these f indings strongly suggest that the increased rate of Bcl-x L deamidation that occurs in response to DNA damage is stimulated by the NHE1-mediated increase in cytosolic pH (Fig.…”

“…Deamidation of Bcl-x L decreases cellular Bcl-x L antiapoptotic activity [(24); see erratum (25)]. The NHE1-mediated early increase in cytosolic pH that occurs in DNA damage-induced and other forms of apoptosis (26)(27)(28)(29)(30) mediates the increased rate of Bcl-x L deamidation (31). The mechanism by which DNA damage activates NHE1 is unknown at present.…”