Inorganic Cation Dependence of Putrescine and Spermidine Transport in Human Breast Cancer Cells

Abstract

The mechanism of polyamine uptake in mammalian cells is still poorly understood. The role of inorganic cations in polyamine transport was investigated in ZR-75-1 human breast cancer cells. Although strongly temperature dependent, neither putrescine nor spermidine uptake was mediated by a Na+ cotransport mechanism. In fact, Na+ and cholinium competitively inhibited putrescine uptake relative to that measured in a sucrose-based medium. On the other hand, ouabain, H+, Na+, and Ca2+ ionophores, as well as dissipation of the K+ diffusion potential, strongly inhibited polyamine uptake in keeping with a major role of membrane potential in that process. Polyamine transport was inversely dependent on ambient osmolality at near physiological values. Putrescine transport was inhibited by 70% by decreasing extracellular pH from 7.2 to 6.2, whereas spermidine uptake had a more acidic optimum. Deletion of extracellular Ca2+ inhibited putrescine uptake more strongly than chelation of intracellular Ca2+. In fact, bound divalent cations were absolutely required for polyamine transport, as shown after brief chelation of the cell monolayers with EDTA. Either Mn2+, Ca2+, or Mg2+ sustained putrescine uptake activity with high potency (Km = 50-300 microM). Mn2+ was a much stronger activator of spermidine than putrescine uptake, suggesting a specific role for this metal in polyamine transport. Other transition metals (Co2+, Ni2+, Cu2+, and Zn2+) were mixed activators/antagonists of carrier activity, while Sr2+ and Ba2+ were very weak agonists, while not interfering with Ca2+/Mg(2+)-dependent transport. Thus, polyamine uptake in human breast tumor cells is negatively affected by ionic strength and osmolality, and is driven, at least in part, by the membrane potential, but not by the Na+ electrochemical gradient. Moreover, the polyamine carrier, or a tightly coupled accessory component, appears to have a high-affinity binding site for divalent cations, which is essential for the uptake mechanism.