AJP - Cell Physiology, Vol 262, Issue 6 C1356-C1363, Copyright © 1992 by American Physiological Society
Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells
G. E. Groblewski, P. T. Hargittai and E. R. Seidel
Department of Physiology, School of Medicine, East Carolina University, Greenville, North Carolina 27858.
Regulation of putrescine uptake in a small intestinal crypt cell line,
IEC-6 cells, was examined. Uptake of [14C]putrescine was measured
throughout a normal growth curve and was found to be inversely related to
growth. Kinetic analysis at low and high cell density revealed the
inhibition of uptake in confluent cells was due to a five-fold reduction in
Vmax of uptake, 199.5 vs. 43.1 pmol.10(5) cells-1.h-1, respectively. Three
gastrointestinal hormones, gastrin, secretin, and cholecystokinin, produced
partial inhibition of [14C]putrescine uptake. Conversely, treatment of
quiescent cells with 5% fetal bovine serum to stimulate growth did not
affect uptake. Influence of putrescine uptake on free ionized intracellular
Ca2+ ([Ca2+]i) was measured by microspectrofluorometry using the
Ca(2+)-sensitive fluoroprobe fura-2. Basal [Ca2+]i was calculated to be 112
nM and increased rapidly to 313 nM upon addition of 10 microM putrescine.
Preventing the rise in [Ca2+]i using an intracellular Ca2+ buffer,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl
ester, decreased [14C]putrescine uptake to 29.5 +/- 5.3% of control values.
45Ca2+ flux experiments and measurement of transport in 0 Ca2+ and 0.5 mM
EDTA suggested an intracellular source of calcium was mobilized during
putrescine uptake. Finally, use of the putative calmodulin antagonist
N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide caused a dose-dependent
inhibition of [14C]putrescine uptake with 50% inhibitory concentration of
approximately 7 microM. These data suggest that putrescine uptake in IEC-6
cells may be regulated by a Ca2+/calmodulin-dependent mechanism.
