Amiloride inhibits murine erythroleukemia cell differentiation: Evidence for a Ca²⁺ requirement for commitment

The effect of amiloride (an inhibitor of passive Na⁺ transport in many tissues) on the differentiation of murine erythroleukemia cells was investigated. Amiloride completely blocked the dimethyl sulfoxide (Me₂SO)-induced erythroid differentiation of cells at a concentration (10 μg/ml) that did not affect cell proliferation. Amiloride also prevented the decrease in cell volume normally observed after a 20-hr exposure to Me₂SO. The ratio of total cell Na⁺ to total cell water was essentially the same for control cells, Me₂SO-treated cells, and cells treated with Me₂SO plus amiloride. However, cells treated for 24 hr with Me₂SO had a rate of Ca²⁺ uptake that was twice that of untreated cells and a similarly higher Ca²⁺ content. Addition of amiloride plus Me₂SO prevented both the increase in Ca²⁺ uptake rate and the increase in Ca²⁺ content. Cells grown in the presence of Me₂SO plus amiloride initiated differentiation immediately after removal of amiloride or addition of the Ca²⁺ ionophore A23187 (1 μg/ml). Addition of sufficient ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce free extracellular Ca²⁺ to submicromolar levels prevented Me₂SO-induced differentiation while only slightly affecting cell proliferation. These results suggest that an increase in the Ca²⁺ level is an essential step in Me₂SO induction, that amiloride either directly or indirectly inhibits this process, and that Me₂SO has an early effect on cells that is necessary for differentiation and is not mimicked by A23187.