TRANSPORT AND STEADY-STATE ACCUMULATION OF PUTRESCINE IN BRUSH-BORDER MEMBRANE-VESICLES OF RABBIT SMALL-INTESTINE

Absorption of polyamines from the lumen is essential for cell proliferation in small intestine but also in other rapidly growing body tissues and tumors. Intestinal uptake of polyamines is thought to involve one or more transport systems, but the characteristics of these systems have not yet been clearly elucidated. Because high levels of putrescine have been identified in intestinal lumen, we explored kinetic, physicochemical, and structural features of uptake of this diamine across rabbit intestinal brush-border membrane vesicles (IBBMV) prepared by CaCl2 or MgCl2 precipitation procedure. Initial rates of putrescine influx were measured during 5-min incubations at 25 or 37 degrees C (optimal temperature) for concentrations of 0.45-145 mu M. At both temperatures, kinetics of putrescine transport fitted a model with a single Michaelis-Menten uptake component plus a nonsaturable uptake component. At 37 degrees C, the kinetic parameters for the saturable component of putrescine uptake, K-m,K-app and V-max,V-app, were 16.8 +/- 4.7 mu M and 19.9 +/- 2.8 pmol . mg protein(-1). min(-1), respectively. The value of the constant for the nonsaturable component of putrescine uptake (P = 0.45 +/- 0.06 x 10(-8) 1 . mg protein(-1). s(-1)) suggested this component represented essentially nonspecific binding of putrescine to IBBMV. Cadaverine, spermidine, and spermine were competitive inhibitors of putrescine transport, with inhibition constants equal to 47, 117, and 219 mu M, respectively. When effects of a variety of alkyldiamines and structural analogues of polyamines (1 mM) on influx of 5.6 mu M putrescine were compared, cadaverine, methylglyoxal bis(guanylhydrazone) (MGBG), and cyclic derivatives of MGBG were found to exhibit the highest inhibitory potencies. Steady-state uptake of putrescine at 25 degrees C was much greater than could be explained by equilibration of medium and intravesicular concentrations of the diamine. Steady-state uptake fitted a single-site model that saturated in the micromolar range. Reversibility of steady-state binding of putrescine was observed after [H-3]putrescine-preloaded IBBMV were diluted 167-fold in incubation buffer at 25 degrees C. Putrescine uptake was also reversed when incubation temperature was raised from 25 to 37 degrees C. In this case, however, degradation of putrescine occurred; a radioactive putrescine by-product was released into the extravesicular medium. Preincubating IBBMV with membrane-impermeant 1,2-bis(2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid (10 mM) evoked a dramatic decrease in both influx and steady-state uptake of putrescine, suggesting that extravesicularly bound Ca2+ or Mg2+ is required for these processes. These data indicate that intestinal uptake of putrescine involves a single substrate-selective transport system and that binding to the brush-border membrane may play a role in regulation of intracellular levels of putrescine.