Characterization of a polyamine transport system in murine embryonic palate mesenchymal cells

Abstract

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of developing systems. Ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, has been shown to be causally related to an increase in glycosaminoglycan synthesis in murine embryonic palatal mesenchyme cells (MEPM). In order to understand other mechanisms that exist to regulate polyamine levels in cells derived from the developing craniofacial area, the present study investigated the capacity of MEPM cells to accumulate exogenous putrescine and tests the hypothesis that polyamine transport can serve as an adaptational response of MEPM cells to a change in their ability to synthesize polyamines. Transport was initiated in confluent cultures of MEPM cells by the addition of 0.1 μCi/ml of ¹⁴C-putrescine. The rate of transport, monitored for 20-120 minutes, was found to be a time-dependent saturable process. The rate of initial transport, determined by incubating MEPM cells for 15 minutes in the presence of different concentrations (1.0-20.0 μM) of ¹⁴C-putrescine, was also found to be saturable, suggesting a carrier-mediated event. Lineweaver-Burk analysis of these data revealed an apparent K_m of 5.78 μM and a V_max of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 deg;C or in the presence of 2-4 DNP was dramatically inhibited. Thus, putrescine transport is an active process, dependent upon metabolic energy. Conditions in which (1) NaCl was iso-osmotically replaced with choline chloride or (2) the Na⁺−electrochemical gradient was dissipated with Na⁺, K⁺−specific ionophores resulted in a decreased rate of transport indicating that putrescine transport in these cells is Na⁺ dependent. Noncompetitive inhibition assays utilizing sulfhydryl reagents that blocked sulfhydryl groups inhibited putrescine transport, suggesting that sulfhydryl groups are important for putrescine uptake. Competitive inhibition assays demonstrated that while spermidine and spermine inhibited putrescine uptake, ornithine did not inhibit transport. Spermidine, spermine, and putrescine thus appear to share a common transport system that is separate from that for ornithine. Putrescine transport is subject to adaptive regulation in both exponentially growing and confluent cultures of MEPM cells. DFMO, an irreversible inhibitor of ODC, which depletes endogenous pools of putrescine, stimulated putrescine uptake with an apparent K_m and V_max of 12.58 μM and 3.51 nmol/mg protein/ 15 minutes, respectively, while preloading MEPM cells with putrescine inhibited uptake. The ability of exogenous putrescine to reverse the inhibition of MEPM cellular proliferation by DFMO suggests that MEPM cells can utilize putrescine transport to regulate cellular proliferation. These data indicate that MEPM cells have the capacity to regulate intracellular polyamine levels in an alternative and/or adjunctive manner to de novo synthesis, via transportation of extracellular polyamines. Furthermore, the rate of putrescine transport apears to be adaptively regulated by the intracellular content of polyamines, which may contribute to the homeostatic regulation of cellular polyamine levels and cellular proliferation.