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 microCi/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 microM) of 14C-putrescine, was also found to be saturable, suggesting a carrier-mediated event. Lineweaver-Burk analysis of these data revealed an apparent Km of 5.78 microM and a Vmax of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 degrees 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular characterization of a human cation‐Cl− cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport

“…From initial characterizations, we found that PA transport activity in stable HEK huCCC9a (measured through influx or uptake studies with radioactive PAs) was stimulated after 20-min incubations in a Ca 2þ -free medium (R 0Ca ), consistent with the observation that the activity of certain PA transporters is Ca 2þ -sensitive (Davis and Ristow, 1988;Gawel-Thompson and Greene, 1988;Seiler et al, 1996;Hasne and Ullman, 2005 …”

“…They are as follows: (1) PA influxes attributed to huCCC9a were in the range ''pmol/min/ 10 7 cells or mg proteins,'' half-maximal with extracellular (PA) in the mM range, linear <4 min and unaffected by the [Ca 2þ ] at which the tracer-added media was set. (2) The activity of certain PA transporters is sensitive to changes in intracellular [Ca 2þ ] ( Davis and Ristow, 1988;Gawel-Thompson and Greene, 1988;Seiler et al, 1996;Hasne and Ullman, 2005) as was probably the case for huCCC9a based on the protocol exploited. (3) When other CCCs such as NKCC1, KCC1, and KCC2 are overexpressed in HEK-293 cells, they also accumulate in intracellular compartments but can still reach the surface (as was also demonstrated for huCCC9a in this work) to promote furosemide-sensitive CCC (Isenring et al, 1998;Caron et al, 2000;personal observations).…”

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

“…Ca 2ϩ is also known to affect polyamine transport into eukaryotes, although the effects are disparate. Ca 2ϩ stimulates polyamine uptake in human fibroblasts (48) and breast cancer cells (47), whereas the divalent cation inhibits polyamine transport into embryonic mesenchymal cells (49) and Neurospora crassa (50). Because the calcium ionophore A23687 inhibited putrescine uptake into TUB::HD309-LmPOT1 T. brucei, it is possible that putrescine transport via LmPOT1 is Ca 2ϩ -dependent.…”

Photoaffinity Labeling of a Cell Surface Polyamine Binding Protein

“…Cell proliferation is critically dependent on a constant supply of the intracellular polyamines: putrescine, spermidine (Spd), 1 and spermine (Spm). Under in vivo conditions, this supply is thought to be maintained by sensitively regulated processes involving polyamine biosynthesis, catabolism, and transport.…”

“…From kinetic studies, polyamine transport is known to be an active process that is energy-and temperature-dependent and adheres to Michaelis-Menten kinetics (1)(2)(3). Although the transporter is highly specific for polyamines, various synthetic molecules such as polyamine analogs, methylglyoxal-bis(guanylhydrazone) (MGBG), and paraquat utilize the carrier as a primary means for gaining entry into cells (4).…”