Transport and steady-state accumulation of putrescine in brush
-border membrane vesicles of rabbit small intestine.
Brachet, Patrick, Hajer Debbabi, and Daniel Tome.
Unit[acute]e de Nutrition Humaine et Physiologie Intestinale de
l'I.N.R.A., Facult[acute]e des Sciences Pharmaceutiques et
Biologiques, 4 Avenue de l'Observatoire, 75006 - Paris, France
APStracts 2:0114G, 1995.
Absorption of polyamines from the lumen is essential for cell
proliferation in the small intestine, but also in other rapidly
growing body tissues and in tumors. Intestinal uptake of polyamines
is thought to involve one or more transport system(s), but the
characteristics of this (or these) system(s) are far from being
clearly elucidated. Since high levels of putrescine have been
identified in the intestinal lumen, we explored the kinetic, physico
-chemical and structural features of uptake of this diamine across
rabbit intestinal brush-border membrane vesicles (IBBMV) prepared by
a 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 ranging from 0.45
to 145 [mu]M. At both temperatures, kinetics of putrescine transport
fit a model with a single Michaelis-Menten uptake component plus a
non-saturable uptake component. At 37 degrees C, the kinetic
parameters for the saturable component of putrescine uptake, Kmapp
and Vmaxapp, 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 non
-saturable component of putrescine uptake (P = 0.45 +/- 0.06 10-8 l.mg
protein-1.sec-1) suggested that this component represented
essentially non-specific binding of putrescine to IBBMV. Cadaverine,
spermidine and spermine were competive inhibitors of putrescine
transport, with Kis equal to 47, 117 and 219 [mu]M, respectively.
Comparing the effects of a variety of alkyldiamines and structural
analogues of polyamines (1 mM) on influx of 5.6 [mu]M putrescine,
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 very
much greater than could be explained by equilibration of medium and
intravesicular concentrations of the diamine. Steady-state uptake fit
a single site model which saturated in the micromolar range.
Reversibility of steady-state binding of putrescine was observed
after diluting [3H]putrescine-preloaded IBBMV 167-fold in incubation
buffer at 25 degrees C. Putrescine uptake could also be reversed by
raising the incubation temperature from 25 degrees C to 37 degrees C.
In this case, however, degradation of putrescine occurred; a
radioactive putrescine by-product was released into the
extravesicular medium. Pre-incubating IBBMV with a 10 mM
concentration of membrane impermeant BAPTA evoked a dramatic decrease
in both influx and steady-state uptake of putrescine, suggesting that
extravesicularly-bound Ca2+ or Mg2+ are 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 the regulation of
intracellular levels of putrescine.
Received 3 November 1994; accepted in final form 26 May 1995.
APS Manuscript Number G440-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 July 1995.