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Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic
Abstract:
When grown in medium containing 5 mM potassium and 140 mM sodium, HL-60, a human promyelocytic cell line, maintained a steady-state intracellular K+ concentration of 145 mmol/L cells and a steady-state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 X 10(-8) M. This ouabain-sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half-time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL-60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 microns 3 to 500 microns 3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain-sensitive component of K+ influx fell with a half-time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half-time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.
Insights
Dimethyl sulfoxide (DMSO) induces HL-60 cell differentiation into granulocytes by rapidly reducing potassium (K+) and sodium (Na+) flux. This ion flux modulation may control differentiation events.
Area of Science:
- Cell Biology
- Biochemistry
- Physiology
Background:
- HL-60 cells are a human promyelocytic cell line.
- These cells can be induced to differentiate into granulocytes using dimethyl sulfoxide (DMSO).
- Intracellular ion concentrations, particularly potassium (K+) and sodium (Na+), are crucial for cell function.
Purpose of the Study:
- To investigate the role of ion flux in DMSO-induced differentiation of HL-60 cells.
- To characterize the components of K+ influx and efflux in HL-60 cells.
- To determine how K+ and Na+ flux changes during granulocytic differentiation.
Main Methods:
- Measurement of unidirectional 42K+ influx and efflux in HL-60 cells.
- Inhibition studies using ouabain and furosemide to identify K+ transport mechanisms.
- Induction of differentiation using 1.5% DMSO and monitoring of cell volume, morphology, and ion flux over 6 days.
Main Results:
- HL-60 cells maintained steady-state intracellular K+ (145 mmol/L) and Na+ (30 mmol/L) concentrations.
- Ouabain-sensitive K+ influx was significantly reduced upon DMSO addition, with a half-time of 11 hours.
- K+ efflux rate constant also decreased with a half-time of 14 hours, indicating coordinated ion flux reduction during differentiation.
Conclusions:
- DMSO-induced differentiation of HL-60 cells is associated with rapid and coordinated reductions in both K+ influx and efflux.
- These ion flux changes occur early in the differentiation process and may play a regulatory role.
- The findings suggest that modulation of ion transport is a key event in granulocytic differentiation.