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The effect of ionophores and related agents on the induction of doming in a rat mammary epithelial cell line

Addition of dimethyl sulfoxide (DMSO) and the mammotropic hormones prolactin, hydrocortisone, insulin, and estradiol to confluent cultures of the epithelial cell line Rat Mammary (Rama) 25 increases dramatically the formation of domes in the cell monolayer after 48–72 hr. Associated with the increase in doming is an increase of 24% in the activity of the Na+/K+ ATPase. Both Ca2+ (A23187) and Na+ (monensin, gramicidin J, melittin) ionophores can replace DMSO in inducing domes whilst the K+ ionophore valinomycin inhibits doming. However, there are no synergistic nor additive effects, respectively, with suboptimal or optimal concentrations of A23187 and melittin together. Ouabain, at concentrations which inhibit the Na/K ATPase in vitro, and amiloride, at concentrations reported to inhibit the passive transport of Na+, both inhibit completely the formation of domes induced by DMSO, A23187, and melittin. EGTA, however, inhibits only the induction of doming by DMSO and A23187; it is without effect with melittin. A23187 and melittin induce the major polypeptide changes that occur in doming cultures with DMSO, and most of these changes are also inhibited with ouabain. It is suggested that one possible interpretation of the findings is that the induction of doming by DMSO in Rama 25 cells occurs by means of sequential increases in Ca2+ and Na+ influxes into the cell, and that the increased intracellular concentration of Na+ so produced stimulates the Na+/K+ ATPase, with a net effect of pumping liquid beneath the cellular monolayer.