Abstract
The Na+ and Ca2+ transport properties of cultured murine erythroleukemia (MEL) cells have been investigated. We have previously shown that amiloride prevents dimethyl sulfoxide-induced MEL cell differentiation via inhibition of an essential Ca2+ influx (levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5948-5952). Here we show that external Na+ inhibits Ca2+ influx and stimulates Ca2+ efflux from uninduced MEL cells. Increasing the internal Na+ concentration by a brief incubation of cells with ouabain stimulates the rate of 45Ca2+ influx. Amiloride (40 microM) completely blocks the external Na+-stimulated 45Ca2+ efflux and external Na+-inhibitable 45Ca2+ influx. The same concentration of amiloride had no significant effect on net Na+ uptake. These results suggest that a significant fraction of Ca2+ flux across the MEL cell plasma membrane occurs via a Na+/Ca2+ antiport system and that amiloride prevents differentiation by blocking Ca2+ influx through this system. The importance of a Na+/Ca2+ antiport system for MEL cell differentiation is supported by the following observation: increasing the cellular Na+ level by a brief treatment with ouabain plus monensin accelerates MEL cell commitment as effectively as adding the Ca2+ ionophore A23187. We suggest that dimethyl sulfoxide induces MEL cell differentiation by inhibiting the Na+ pump and consequently allowing Ca2+ influx through the Na+/Ca2+ antiport.
Highlights
The impor- (Na,K)-ATPase inhibitor ouabain induced erythroid differentiation at ferentiation is supported by the following observation: concentrations which caused 20 to 40% inhibition of active increasing the cellular Na+ level by a brief treatment potassium uptake [16].These results suggest that alterations with ouabain plus monensin accelerates MEL cell com- in cellular Na’ and K+ maybe essential for commitment
A23187.We suggest that dimethyl sulfoxide induces MEL cell differentiation by inhibiting the Na’ pump and allowing Ca2+ influx throughthe Na+/Ca2+antiport
We have found that an increase in cellular Ca2+is essential for terminal differentiation of MEL cells based on studies with the drug amiloride [5]
Summary
Cell Lines and Culture Conditions-Cell line 745 was originally obtained from Dr C. The trapped volume in the pellet was determined by adding either [I4C]sucroseor [3H]waterto cell suspension samples (in duplicate) and centrifuging through oil as described above. The cells were incubated at 37"C and 1.0-ml samples were removed periodically, centrifuged through oil as described above, and the pellets counted for 14C.In a second experiment, the cells were incubated on ice, in Na+-Ringer's buffer. Samples (0.2 m l ) were removed a t intervals, diluted into 0.8 ml of choline-Ringer's (0 "C) and rapidly centrifuged through oil as described above, to separate the cells from the buffer. Samples (1.0 m l ) were withdrawn at intervals and treated as described above to separate the cells from medium and to count for *"Rb
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