Characterization of univectorial polyamine transport in duodenal crypt cell line

Abstract

High levels of polyamines have been identified in the lumen of the intestines. Luminal polyamines are involved in normal mucosal growth and may be the primary source of extracellular polyamines for tumors grown in animals undergoing polyamine antimetabolite therapy. The vectorial movement of polyamines across an in vitro model of the gut was studied in epithelial cells grown in culture. IEC-6 cells were plated on either plastic or raised inserts. Cells grown on plastic were employed to define the kinetic constants for putrescine and spermidine uptake. Eadie-Hofstee plot analysis of putrescine uptake was characteristic of a single class of transporter with a Michaelis constant (Km) of 4.85 +/- 0.57 microM and a maximal velocity (Vmax) of 627 +/- 85 pmol x 15 min-1 x 10(6) cells-1. The plot for spermidine uptake was curvilinear and representative of the interaction of spermidine with two sites: Km 1 and 2 are 0.26 +/- 0.13 and 2.1 +/- 0.77 microM; Vmax 1 and 2 are 177 +/- 50 and 429.5 +/- 70 pmol x 15 min-1 x 10(6) cells-1, respectively. Calmodulin antagonism blocked the uptake of putrescine and the low-affinity but not high-affinity spermidine uptake system. Seven-day postconfluent cells grown on plastic inserts were used to study the vectorial movement of polyamines in a polarized epithelium. The apical membrane domain expressed two sites with similar kinetic constants to those observed when cells were grown on plastic. In contrast, however, the basolateral membrane did not transport polyamines. Spermidine uptake through this membrane was only a fraction of that in the apical membrane and was completely nonspecific.(ABSTRACT TRUNCATED AT 250 WORDS)