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

Absorption of polyamines from the lumen is essential for cell prolifer ation in small intestine but also in other rapidly growing body tissue s and tumors. Intestinal uptake of polyamines is thought to involve on e or more transport systems, but the characteristics of these systems have not yet been clearly elucidated. Because high levels of putrescin e have been identified in intestinal lumen, we explored kinetic, physi cochemical, and structural features of uptake of this diamine across r abbit intestinal brush-border membrane vesicles (IBBMV) prepared by Ca Cl2 or MgCl2 precipitation procedure. Initial rates of putrescine infl ux were measured during 5-min incubations at 25 or 37 degrees C (optim al temperature) for concentrations of 0.45-145 mu M. At both temperatu res, kinetics of putrescine transport fitted a model with a single Mic haelis-Menten uptake component plus a nonsaturable uptake component. A t 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 a nd 19.9 +/- 2.8 pmol . mg protein(-1). min(-1), respectively. The valu e 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 inhibito rs of putrescine transport, with inhibition constants equal to 47, 117 , and 219 mu M, respectively. When effects of a variety of alkyldiamin es and structural analogues of polyamines (1 mM) on influx of 5.6 mu M putrescine were compared, cadaverine, methylglyoxal bis(guanylhydrazo ne) (MGBG), and cyclic derivatives of MGBG were found to exhibit the h ighest inhibitory potencies. Steady-state uptake of putrescine at 25 d egrees 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 ran ge. Reversibility of steady-state binding of putrescine was observed a fter [H-3]putrescine-preloaded IBBMV were diluted 167-fold in incubati on buffer at 25 degrees C. Putrescine uptake was also reversed when in cubation 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 extravesicularl y bound Ca2+ or Mg2+ is required for these processes. These data indic ate that intestinal uptake of putrescine involves a single substrate-s elective transport system and that binding to the brush-border membran e may play a role in regulation of intracellular levels of putrescine.