CONTROL OF INTRACELLULAR PH DURING REGULATORY VOLUME DECREASE IN HL-60 CELLS

Intracellular pH (pH(i)) homeostasis was investigated in human promyel ocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pH(i) is regulated and which transport systems are involved. Cells suspended in hypotonic (5 0-60% of isotonic) media undergo a small (<0.2 pH units), but signific ant (P < 0.05), intracellular acidification within 5 min. However, aft er 30 min of RVD, pH(i) is not significantly different from the initia l pH(i) in 20 mM HCO3- medium and is significantly higher in HCO3--fre e medium. Experiments performed in media with or without 150 mu M 4,4' -diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate tha t the anion exchanger (AE) mediates a net Cl- influx, with compensatin g HCO3- efflux, during RVD. To determine which transport systems are i nvolved in counteracting this tendency toward acidification, we measur ed transport rates and examined the effect of transport system inhibit ors on pH(i). We found that inhibition of Na+/H+ exchange (NHE) with 1 2.5 mu M ethylisopropylamiloride (EIPA) causes pH(i) to fall significa ntly by the end of 30 min of RVD. As assessed by EIPA-sensitive Na-22( +) uptake measurements, NHE, largely dormant under resting isotonic co nditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pH(i) and cell volume to near-normal levels. Thus a shift in the normal pH(i) dependence and/or volume dependence of NH E activity must occur during RVD under hypotonic conditions. In contra st, H+-monocarboxylate cotransport appears to play only a supportive r ole in pH regulation during RVD, as indicated by lack of stimulation o f [C-14]lactate efflux during RVD.