Control of intracellular pH during regulatory volume decrease in HL-60 cells

Intracellular pH (pH(i)) homeostasis was investigated in human promyelocytic 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 (50-60% of isotonic) media undergo a small (<0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pH(i) is not significantly different from the initial pH(i) in 20 mM HCO₃/^- medium and is significantly higher in HCO₃/^--free medium. Experiments performed in media with or without 150 μM 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid and HCO₃/^- demonstrate that the anion exchanger (AE) mediates a net Cl^- influx, with compensating HCO₃ efflux during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pH(i). We found that inhibition of Na⁺/ H⁺ exchange (NHE) with 12.5 μM ethylisopropylamiloride (EIPA) causes pH(i) to fall significantly by the end of 30 min of RVD. As assessed by EIPA sensitive ²²Na⁺ uptake measurements, NHE, largely dormant under resting isotonic conditions, 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 NHE activity must occur during RVD under hypotonic conditions. In contrast, H⁺-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [¹⁴C]lactate efflux during RVD.