(Received for publication, August 29, 1994; and in revised form, November 14, 1994)
From the
The mechanism of polyamine uptake in mammalian cells is still
poorly understood. The role of inorganic cations in polyamine transport
was investigated in ZR-75-1 human breast cancer cells. Although
strongly temperature dependent, neither putrescine nor spermidine
uptake was mediated by a Na cotransport mechanism. In
fact, Na
and cholinium competitively inhibited
putrescine uptake relative to that measured in a sucrose-based medium.
On the other hand, ouabain, H
, Na
,
and Ca
ionophores, as well as dissipation of the
K
diffusion potential, strongly inhibited polyamine
uptake in keeping with a major role of membrane potential in that
process. Polyamine transport was inversely dependent on ambient
osmolality at near physiological values. Putrescine transport was
inhibited by 70% by decreasing extracellular pH from 7.2 to 6.2,
whereas spermidine uptake had a more acidic optimum. Deletion of
extracellular Ca
inhibited putrescine uptake more
strongly than chelation of intracellular Ca
. In fact,
bound divalent cations were absolutely required for polyamine
transport, as shown after brief chelation of the cell monolayers with
EDTA. Either Mn
, Ca
, or
Mg
sustained putrescine uptake activity with high
potency (K
= 50-300
µM). Mn
was a much stronger activator of
spermidine than putrescine uptake, suggesting a specific role for this
metal in polyamine transport. Other transition metals
(Co
, Ni
, Cu
, and
Zn
) were mixed activators/antagonists of carrier
activity, while Sr
and Ba
were very
weak agonists, while not interfering with
Ca
/Mg
-dependent transport. Thus,
polyamine uptake in human breast tumor cells is negatively affected by
ionic strength and osmolality, and is driven, at least in part, by the
membrane potential, but not by the Na
electrochemical
gradient. Moreover, the polyamine carrier, or a tightly coupled
accessory component, appears to have a high-affinity binding site for
divalent cations, which is essential for the uptake mechanism.
In addition to the enzymes required for polyamine biosynthesis, most prokaryotic and eukaryotic cells possess one or several membrane transport activities with a high affinity for natural polyamines(1, 2) . Polyamine uptake activity in mammalian cells becomes dramatically elevated upon the addition of mitogens or hormones (2, 3, 4, 5) and after cell transformation(2, 6) . Furthermore, transport of plasma polyamines derived from various sources, including enterohepatic circulation(7) , has been identified as a major mechanism through which tumor cells can compensate polyamine depletion induced by specific enzyme inhibitors (8, 9) .
Several classes of high affinity polyamine carriers have recently been identified and cloned in Escherichia coli(10, 11, 12, 13) . Among these, spermidine (11) and putrescine preferential (13) carriers are respectively encoded by different regulons made of four separate genes with primary structures characteristic of ATP-binding cassette transporters(14) . As expected for such bacterial transporters(14) , putrescine and polyamine uptake in E. coli is stringently energy dependent(15, 16) , and depends on periplasmic binding proteins with high affinity for the specific substrates(11, 13, 16) . However, unlike uptake by other ATP-binding cassette transporters(14) , polyamine transport in bacteria also requires a protonmotive membrane potential(15, 16) .
On the other hand, membrane carriers responsible for polyamine transport in eukaryotes have not yet been characterized at the biochemical or molecular levels. The physiological characterization of polyamine uptake has been carried out in a variety of cell types (for review, see (2) , 17) with considerable divergence with respect to the general properties and mechanism of the transport system(s) involved. There is general agreement on the marked energy dependence of the polyamine uptake process in mammalian cells(2, 17) , but the nature of the energy coupling mechanism is as yet unclear. Much uncertainty exists on the electrochemical driving force, the stoichiometric characteristics, as well as the number of carrier species involved in the di- and polyamine uptake process.
For instance, a substantial
fraction of total putrescine uptake in intact mammalian cells has been
reported to require extracellular Na ([Na
]
), (
)while spermidine and spermine transport is rather
insensitive to [Na
]
deletion (rev. in 2, 18). On the other hand, no evidence for
a [Na
]
requirement has
been found for either putrescine or polyamine uptake in other cell
types(2, 7, 18, 19, 20, 21) or
membrane vesicles(22) . Evidence for a
[Na
]
dependence for
polyamine uptake has been derived either from substitution experiments
with other electrolytes such as choline chloride or LiCl (e.g. 18, 23, 24) or from the inhibition caused by ionophores,
Na
channel blockers, or
ouabain(23, 25, 26) . However, other workers
have postulated that the main driving force for polyamine uptake in
eukaryotic cells is an electronegative membrane potential, as in bovine
lymphocytes (27) or yeast vacuoles(28) .
A possible
role for other ionic factors such as H,
K
, Ca
, or Mg
in
the mechanism of polyamine internalization has received much less
attention. Intracellular Ca
([Ca
]
) has been
proposed to regulate polyamine transport, based on its inhibition by
calmodulin
antagonists(28, 29, 30, 31) , on the
elevation of [Ca
]
observed concomitantly with ongoing uptake (30, 31, 32) and on the decreased uptake
activity brought about by chelation of
[Ca
]
(30, 31) .
The source for the increase in
[Ca
]
observed upon
putrescine addition was proposed to be intracellular
stores(30, 31) , although earlier observations in
human fibroblasts had suggested that extracellular Ca
([Ca
]
) and
Mg
have marked and rapid effects on putrescine
transport (33) . Adding to the complexity of the role of
Ca
in polyamine uptake,
[Ca
]
strongly inhibits
a low-affinity, saturable putrescine transport system in Neurospora
crassa, and a mutation has been identified in this fungus that
constitutively relieves this inhibition(34) .
In the companion article(5) , we have characterized the kinetic properties, hormonal regulation and the mechanism of feedback inhibition of a highly active transport system for putrescine and spermidine uptake in estrogen-responsive ZR-75-1 human breast cancer cells. Kinetic analysis shows that putrescine and spermidine transport in ZR-75-1 cells is either mediated by a single class of carrier or by dual but very similar agencies exclusive for putrescine and polyamines, respectively, with extensive mutual inhibition by heterologous substrates(5) .
In order to
define the mechanistic determinants of polyamine internalization in
this cell line, we have systematically assessed the role of various
ionic parameters in the uptake process. We are reporting that neither
putrescine and spermidine uptake in ZR-75-1 cells is a
Na cotransport process, but is in fact competitively
inhibited by high cation concentrations. Polyamine uptake is inversely
regulated by ambient osmolality and exhibits a well-defined dependence
on extracellular pH, putrescine, and spermidine having markedly
different pH optima as substrates. The transport process is highly
sensitive to experimental maneuvers that decrease the membrane
potential. Furthermore, we provide novel evidence that polyamine uptake
in human breast cancer cells has an absolute, high affinity requirement
for extracellular divalent metals such as Ca
,
Mg
, and Mn
, suggesting that the
carrier has a tight binding site for such cations which is essential
for its activity.
To study the dependence
of putrescine and polyamine uptake on
[Na]
, NaCl (103 mM) was
deleted from the basic formulation and added at various concentrations,
using sucrose or choline chloride to achieve the final osmolality of
complete RPMI 1640 medium (325 mosmol/kg). In some experiments,
NaHCO
(23.8 mM) and Na
HPO
(5.6 mM) were also deleted from the basic formulation
and isosmotically replaced with sucrose to obtain a nominally
Na
-free medium. The effect of osmolality was similarly
assessed by varying the NaCl concentration without osmotic replacement
or by selectively deleting NaCl from the basic formulation and adding
increasing amounts of sucrose to achieve the desired final osmolality,
all other constituents being kept constant. Osmolality was measured by
cryoscopy with a freezing point depression osmometer (Advanced
Instruments)(36) . The effect of extracellular K
([K
]
) was studied by
adding KCl (normally at 5.4 mM) at the desired concentration
to an initially NaCl- and KCl-free RPMI 1640-based medium, and NaCl was
added to obtain a constant total NaCl + KCl concentration (108.4
meq of Cl
). The effect of Ca
,
Mg
, and other divalent metals was studied by
selectively deleting CaCl
and/or MgSO
(each
normally at 0.4 mM) from the basic medium formulation, with
subsequent addition of the appropriate metal salt to the desired final
concentration. Where indicated, cell monolayers were briefly rinsed
with 1 ml of serum- and Ca
/Mg
-free
RPMI 1640 medium containing 0.5 or 1 mM EDTA prior to uptake
assays.
The effect of extracellular pH was measured in serum-, amino
acid- and NaHCO-free RPMI 1640 medium buffered with 10
mM Tris, 10 mM MOPS. The desired pH value (at 37
°C) was obtained by adding a known amount of HCl (for pH < 7.4)
or NaOH (for pH > 7.4). As a reference, uptake activity was measured
in parallel cell cultures incubated with the same medium buffered at pH
7.4 and supplemented with concentrations of NaCl osmotically equivalent
to the NaOH or HCl added for adjusting each pH value tested.
For the
determination of the kinetic parameters of transport, the substrate
concentration was varied in the respective medium to be tested by
adding increasing concentrations of nonradioactive substrate to a fixed
amount of [H]putrescine and
[
H]spermidine, and the K
and V
values were determined by Lineweaver-Burk
analysis.
Figure 1:
Temperature dependence of putrescine
and spermidine uptake in ZR-75-1 human breast cancer cells. At
time 0, 20 µM [H]putrescine (50
Ci/mol) (
,
) or 5 µM [
H]spermidine (500 Ci/mol) (
,
) was added to ZR-75-1 cell monolayers in HEPES-buffered,
serum-free RPMI 1640, either at 4 (plainsymbols) or
37 °C (solidsymbols), as described under
``Experimental Procedures.'' Intracellular radioactivity was
determined at the indicated incubation periods. Each point is the mean
± S.D. of determinations from triplicate cultures. When no bar
is shown, the experimental deviation was smaller than the symbol
used.
Figure 2:
Effect
of extracellular Na on the kinetic parameters of
putrescine and spermidine uptake. A and B,
ZR-75-1 cells were preincubated for 15 min in serum-free RPMI
1640 medium in which NaCl was isosmotically substituted with either
choline chloride (
) or sucrose (
) to yield the indicated
Na
concentration. [
H]Putrescine (A) and [
H]spermidine uptake (B) was then determined for 20 min under the same experimental
conditions. Each point is the mean ± S.D. of determinations from
triplicate cultures. C, Lineweaver-Burk analysis of
[
H]putrescine uptake in serum-free RPMI 1640
medium containing the normal NaCl concentration (103 mM)
(
), or in which total NaCl was isosmotically replaced with choline
chloride (
) or sucrose (
). Na
HPO
and NaHCO
normally present in RPMI 1640 medium at
23.8 and 5.6 mM, respectively, were isosmotically substituted
in all groups with sucrose (65 mM), in order to obtain
nominally Na
-free conditions for choline chloride- and
sucrose-based media. K
(app), apparent K
values of putrescine transport
determined by Michaelis-Menten analysis in each medium composition
assuming no inhibition of the uptake
process.
In fact, kinetic analysis showed that
choline chloride and NaCl both interfere with putrescine uptake by
decreasing the apparent affinity of the substrate, but not the V, relative to that measured in a sucrose-based
medium (Fig. 2C). Michaelis-Menten analysis for the
behavior of inhibitors was not strictly applicable to the present model
due to the essential osmotic contribution of the interfering compounds.
Nevertheless, assuming that sucrose was essentially inert toward the
putrescine carrier, Fig. 2C suggests that Na
and cholinium ions might be described as competitive inhibitors
of putrescine transport relative to that measured in a sucrose-based
medium, with apparent K
values of 139 and 22
mM, respectively.
Indeed, ouabain, a
specific inhibitor of the plasma membrane
Na/K
-ATPase, inhibited putrescine (Table 1) as well as spermidine uptake (data not shown), in
keeping with other mammalian cells(2, 17) .
Furthermore, the Na
-preferential ionophore gramicidin
D inhibited putrescine uptake by 59% when added to the standard assay
mixture (108 mM NaCl). However, the ionophore had little
effect on putrescine uptake when NaCl was substituted with sucrose. The
K
-selective ionophore valinomycin had virtually no
effect on putrescine uptake, which can most likely be attributed to the
high basal K
conductance of breast epithelial
cells(38) . On the other hand, the protonophore CCCP inhibited
putrescine uptake even more potently than gramicidin D (Table 1).
In human breast cancer cells, membrane potential is primarily
determined by a K diffusion potential(38) .
Therefore, increasing the concentration of extracellular K
([K
]
) should depolarize
the plasma membrane in accordance with the Nernst
equation(38, 39) . We thus examined the effect of
dissipating the negative membrane potential of ZR-75-1 cells by
isosmotically increasing [K
]
at
the expense of [Na
]
. Fig. 3A shows that the rate of putrescine uptake was
indeed strongly decreased in a log-linear fashion when
[K
]
was increased from 1 to 100
mM, as would be expected if the rate of internalization of the
diamine was proportional to the K
diffusion potential.
However, these data would also be consistent with direct inhibition of
carrier activity by K
ions, similar to the effect of
NaCl or choline chloride. The latter possibility was examined by
determining the effect of [K
]
on
the kinetic parameters of putrescine and spermidine uptake. High
[K
]
(50 mM) did not
substantially affect the apparent affinity for putrescine (Fig. 3B) or spermidine uptake (Fig. 3C), but rather selectively decreased the V
. Although Michaelis-Menten kinetics could not
again be formally applied to the present model, high
[K
]
nevertheless acted similarly
to a non-competitive inhibitor of putrescine uptake relative to the
parameters measured under normal ionic conditions. Uptake inhibition by
[K
]
was thus qualitatively
different from that exerted by NaCl and choline chloride, suggesting
that K
did not solely act by direct competition with
the carrier binding site.
Figure 3:
Effect of extracellular K
on the kinetic parameters of putrescine and spermidine uptake. A, concentration dependence of
[
H]putrescine uptake on extracellular
K
. ZR-75-1 cells were preincubated for 10 min in
serum-free RPMI 1640 medium containing the indicated KCl concentration
at constant ionic strength, as described under ``Experimental
Procedures.'' The rate of [
H]putrescine
uptake was then determined for 20 min under the same experimental
conditions. Each point is the mean ± S.D. of determinations from
triplicate cultures. The curve shown represents fitting of the results
with a logarithmic equation. B and C, Lineweaver-Burk
analysis of [
H]putrescine and
[
H]spermidine uptake, respectively, under normal
(103 mM NaCl, 5.4 mM KCl) (
) and high
[K
]
(58.4 mM NaCl, 50 mM KCl) (
)
conditions.
The effect of CCCP and valinomycin on
putrescine and spermidine transport was further evaluated by varying
the transmembrane K gradient (Table 2). The
inhibition of both putrescine and spermidine uptake by high
[K
]
(100 mM KCl) was
larger than that exerted by CCCP, and the protonophore only slightly
increased transport inhibition by high
[K
]
. On the other hand,
valinomycin, while having virtually no effect on the rate of either
putrescine or spermidine uptake at normal or high
[K
]
, partly reversed the
inhibition exerted by CCCP on these parameters at normal
[K
]
.
In E. coli,
putrescine efflux is stimulated by inward K transport
through an osmotically sensitive exchange process(40) .
Conversely, a nonspecific stimulation of K
efflux by
high rates of putrescine uptake has been reported in the fungus N.
crassa(34) . Thus, in order to assess the possibility that
K
might participate in the polyamine uptake mechanism
in a countertransport or cotransport fashion, the effect of spermidine
and putrescine uptake on transmembrane
Rb fluxes was
examined in ZR-75-1 cells. As shown in Fig. 4, neither
Rb influx or efflux was significantly influenced (for up
to 30 and 120 min, respectively) by putrescine or spermidine addition
at concentrations nearly saturating uptake activity(5) . Thus,
neither putrescine or polyamine uptake is detectably coupled to net
changes in K
fluxes, as assessed with the
Rb tracer. These data also show that ouabain had the
expected, rapid inhibitory effect on Na
/K
ATPase activity (Fig. 4A), while
Rb
efflux was markedly accelerated during the initial 10 min following the
removal of Ca
and Mg
(Fig. 4B). The latter effect is consistent with
the activation of Ca
-dependent K
channels upon the expected increase in
[Ca
]
caused by a reversal of
the transmembrane Ca
gradient(41) .
Figure 4:
Effect of exogenous putrescine and
spermidine on Rb transmembrane fluxes. A, time
course of
Rb influx. ZR-75-1 cells were incubated at
room temperature and atmospheric gas composition for the indicated time
in HEPES-buffered, serum-free RPMI 1640 medium containing 3.9 mM
RbCl, and either 20 µM putrescine
(
,
), 10 µM spermidine (10 µM)
(
,
) or no amine addition (
,
), in the presence (solidsymbols) or absence (plainsymbols) of 1 mM ouabain, as described under
``Experimental Procedures.'' B, time course of
Rb efflux. Following preloading of ZR-75-1 cells for
1 h at room temperature with
RbCl, tracer was removed,
cultures transferred to 37 °C, and intracellular radioactivity was
determined at the intervals shown in RPMI 1640 medium supplemented with
20 µM putrescine (
,
), 10 µM spermidine (
,
), or no amine (
,
), in the
presence (plainsymbols) or absence (solidsymbols) of CaCl
and MgSO
(0.42
and 0.41 mM, respectively). Each point is the mean ±
S.D. of determinations from triplicate
cultures.
Figure 5:
Effect of ambient osmolality on putrescine
and spermidine uptake activity. ZR-75-1 cells were incubated for
15 min at the indicated osmolality as adjusted with NaCl () or
sucrose (
), prior to a 20-min assay of
[
H]putrescine (A) and
[
H]spermidine uptake (B) under the same
experimental conditions, as described under ``Experimental
Procedures.'' Data are the mean ± S.D. of determinations
from triplicate cultures.
Figure 6:
Effect of extracellular pH on the rate of
putrescine and spermidine uptake. ZR-75-1 cells were preincubated
for 60 min in control buffer solution (pH 7.4) prior to parallel
determination of [H]putrescine (
) and
[
H]spermidine uptake (
) for a 20-min period
at the indicated pH as described under ``Experimental
Procedures.'' Each point is the mean ± S.D. of
determinations from triplicate cultures, as expressed as percentage of
the uptake determined at pH 7.4 (control).
Figure 7:
Effect of chelation of extracellular or
intracellular Ca, and of calcium ionophore A23187. A, ZR-75-1 cells were incubated for 15 min in serum-free
RPMI 1640 medium containing 420 µM CaCl
(control, containing), or nominally Ca
-free
(-Ca
), in the presence or absence of 1 mM EGTA and/or 10 µM A23187, and
[
H]putrescine uptake was then measured for 20 min
under the same experimental conditions. B, cells were
preloaded with the indicated concentration of BAPTA-AM for 45 min in
Ca
- and Mg
-containing serum-free
RPMI 1640 medium, and then rinsed twice with Ca
-free
medium (containing 0.41 mM MgSO
) plus 2 mM EGTA, before a 20-min assay of [
H]putrescine
uptake in the presence (solidbars) or absence (plainbars) of Ca
. EGTA (2
mM) was added to Ca
-deleted media during the
uptake assay. Each point is the mean ± S.D. of determinations
from triplicate cultures.
In fact,
putrescine uptake in ZR-75-1 cells was directly and completely
dependent on an extracellular source of divalent cations, as shown
after briefly ``stripping'' cell monolayers with 0.5 mM EDTA (Fig. 8). Ca was more active than
Mg
in sustaining putrescine uptake, with EC
values of about 50 and 300 µM respectively, and the
maximal uptake stimulation observed in its presence was about
20-25% higher than with Mg
(Fig. 8A). Omitting the EDTA rinsing step prior
to the uptake assay preserved a basal rate of putrescine transport in
the nominal absence of divalent cations, which was equivalent to about
40% of the value measured at optimal
[Ca
]
. As observed with
Ca
chelators, preincubation in the absence of
Ca
strongly decreased subsequent putrescine uptake
measured upon repletion of the divalent cation, while the rate of
diamine transport was less sensitive to prior incubation in
Mg
-free medium (Fig. 8B). Moreover,
the effect of optimally active concentrations of Ca
and Mg
(800 µM) on putrescine
uptake showed partial additivity when ZR-75-1 cells were
preincubated under Ca
-free conditions, but
Ca
alone could sustain a near maximal rate of
putrescine uptake when only the availability of Mg
was varied during preincubation.
Figure 8:
Dependence of putrescine uptake on
extracellular Ca and Mg
A, concentration dependence of putrescine transport on
extracellular Ca
or Mg
.
ZR-75-1 cells were preincubated for 15 min in serum-free RPMI
1640 containing 0.42 mM CaCl
and 0.41 mM MgSO
, rinsed for 60 s with
Ca
/Mg
-free medium containing 0.5
mM EDTA, and then assayed for
[
H]putrescine uptake during 20 min in RPMI 1640
medium containing the indicated concentration of Ca
(
) or Mg
(
). The dottedcolumn indicates putrescine uptake activity measured in
the absence of divalent cations when no EDTA was added at the rinsing
step. B, cells were incubated for 15 min in serum-free RPMI
1640 containing 0.8 mM CaCl
and/or 0.8 mM MgSO
, rinsed with
Ca
/Mg
-free medium containing 0.5
mM EDTA, and assayed for [
H]putrescine
uptake in the presence or absence of Ca
and/or
Mg
(0.8 mM each), as indicated. Data are
represented as the mean ± S.D. of determinations from triplicate
cultures.
Other divalent cations were
tested for their ability to influence putrescine uptake in
ZR-75-1 cells (Table 3). In the complete assay medium, all
transition metals tested except Mn had some ability
to inhibit Ca
/Mg
-stimulated
putrescine uptake at equimolar concentration (800 µM),
Zn
being clearly the most potent in this respect.
Transition metals were also endowed with significant ability to sustain
putrescine uptake in the nominal absence of Ca
and
Mg
, in the order Zn
<
Ni
Cu
< Co
Mg
< Ca
<
Mn
. On the other hand, Sr
and
Ba
(each at 800 µM), which are
high-affinity substrates for Ca
channels(49) , had no effect on
Ca
/Mg
-stimulated putrescine
transport. While Sr
slightly stimulated putrescine
transport, Ba
was virtually inactive.
We next
compared the effect of Ca and Mg
on
putrescine and spermidine uptake, as well as the potential sites of
action of equimolar concentrations Zn
and
Mn
, respectively, the most potent inhibitor and
inducer of putrescine uptake among the metals tested (Table 4).
Spermidine was even more stringently dependent than putrescine on
extracellular Ca
and Mg
for its
internalization in ZR-75-1 cells, with either cation being
equally and almost maximally effective in this respect. Zn
inhibited the individual effect of both Ca
and
Mg
on the uptake process, using either putrescine or
spermidine as substrate. Furthermore, Mn
was a very
efficient substitute for either Ca
or Mg
in sustaining putrescine and spermidine uptake (Table 4).
In fact, spermidine and putrescine transport had a markedly different
dependence on Mn
concentration (Fig. 9).
Mn
activated putrescine uptake up to 80% of the level
obtained with optimal concentrations of Ca
and
Mg
(EC
50 µM), with a
broad optimum between 100 and 500 µM. On the other hand,
the effect of Mn
on spermidine transport was
biphasic, with a maximal activation between 100 and 200 µM which exceeded Ca
/Mg
-dependent
activation by 45%, and a progressive loss of potency at higher
concentrations.
Figure 9:
Activation of putrescine and spermidine
uptake activity by Mn. ZR-75-1 cells were
preincubated for 10 min in
Ca
/Mg
-free RPMI 1640 medium, rinsed
once with the same medium containing 1 mM EDTA, and then
assayed for [
H]putrescine (
) or
[
H]spermidine uptake (
) during 20 min in
Ca
/Mg
-free medium containing the
indicated concentration of MnCl
. Each point is the mean
± S.D. of determinations from triplicate cultures and are
expressed as the percentage of the uptake measured in parallel in
medium supplemented with 0.8 mM each of CaCl
and
MgSO
.
The steep temperature dependence of putrescine and spermidine
uptake in ZR-75-1 human breast cancer cells clearly identifies
polyamine transport as an energy-requiring mechanism as in most other
cell types(2) . Based on the Na dependence
postulated in some models(2, 18) , it has been
proposed that polyamine uptake functions as a Na
cotransport system using the electrochemical Na
gradient as a driving force, with a secondary energy requirement
due to the maintenance of this gradient by
Na
/K
ATPase(17, 26) . Most, if not all earlier
evidence that polyamine, and especially putrescine, transport is a
Na
-dependent process has been derived from experiments
in which NaCl was substituted with electrolytes such as choline
chloride and LiCl(18, 23, 24) . The present
results demonstrate, however, that Na
and cholinium
ions behave as apparent competitive inhibitors of polyamine uptake in
ZR-75-1 cells and that Na
is completely
dispensable for the uptake process as assessed by substitution with a
non-electrolyte. Competitive inhibition of putrescine uptake by various
inorganic cations has been previously reported in the fungus N.
crassa(34) . Likewise, deletion of extracellular
Na
had no effect on either putrescine or spermidine
uptake in mammalian
cells(7, 19, 20, 21, 50) and E. coli(51) when non-electrolytes
such as sucrose or D-mannitol were substituted as osmolytes.
Thus, the Na dependence previously postulated for
putrescine transport may in fact correspond to the greater competitive
inhibition by electrolytes used as osmotica in
Na
-deleted medium formulations, as well as to the
membrane depolarization expected from replacement of certain
Na
salts with the corresponding K
forms (e.g. Refs. 18, 52). Significant competition by
electrolytes for polyamine-carrier interactions is expected from the
known dependence on ionic strength for polyamine binding to
macromolecular anions(53) . Nonspecific interference of high
cation concentrations with polyamine uptake through coulombic
interactions is also consistent with the observation that the apparent
dependence of uptake on [Na
]
(when substituted with cholinium
or
Li
) decreases in the order putrescine > spermidine
> spermine(54) . Reduced interference of ionic strength with
the uptake of increasingly charged substrates, as also found here in
the relative dependence of putrescine and spermidine transport on
Na
substitution ( Fig. 2A and B) is consistent with their relative affinity for the
mammalian transport system(2, 5) , in a manner similar
to polyamine-nucleic acid interactions(55) .
While the
Na electrochemical gradient per se does not
likely provide the coupling mechanism for energizing polyamine
transport in ZR-75-1 cells, the present data are compatible with
a major role of membrane potential in this respect. Thus, various
depolarizing stimuli, including increased
[K
]
,
Na
/K
ATPase inhibition, and net
Na
, H
, or Ca
influx
by ionophores such as gramicidin D, CCCP, and A23187, respectively,
were all found to rapidly and strongly depress uptake activity. Several
findings point to the plasma membrane potential as a major determinant
of the rate of putrescine and spermidine uptake. First, the dependence
of gramicidin-induced inhibition of putrescine uptake on
[Na
]
and the effect of ouabain
support the conclusion that although the electrochemical Na
potential is dispensable for substrate internalization, net
Na
influx can inhibit polyamine transport through
membrane depolarization. Second, the lack of effect of valinomycin on
polyamine uptake at both normal or high
[K
]
strongly suggests that the
mitochondrial potential is not involved in sustaining the uptake
process and that uncoupling of oxidative phosphorylation by valinomycin
or CCCP did not compromise polyamine uptake in short term experiments.
Third, polyamine uptake inhibition by CCCP occurred at normal
[K
]
and at pH 7.4, with very
little further inhibition upon an increase in
[K
]
and was partly reversed by
outward K
transfer caused by valinomycin. Although at
physiological pH, CCCP might initially induce a slight, short-lived
cytosolic acidification, homeostatic mechanisms rapidly restore normal
steady-state pH
in CCCP-treated cells (56) ,
arguing against a decrease in pH
as underlying the effect
of CCCP on polyamine uptake. In fact, polyamine depletion by the
ornithine decarboxylase inhibitor,
-difluoromethylornithine, which
increases the velocity of polyamine uptake(2, 5) ,
decreases steady-state pH
. (
)Finally, the
inverse logarithmic relationship between
[K
]
and the rate of putrescine
uptake, as well as the non-competitive pattern of uptake inhibition due
to increasing [K
]
, suggest that
dissipating the transmembrane K
gradient depresses
uptake activity through membrane depolarization. Recent experiments
with membrane potential probes indeed confirm that the degree of plasma
membrane depolarization is closely correlated with the relative
inhibition of polyamine uptake by ionophores and high
[K
]
. (
)
Although
membrane potential markedly influences the rate of polyamine transport,
it is far less clear how this parameter is actually coupled to the
uptake mechanism. In E. coli cells or membrane vesicles, a
protonmotive potential is clearly required to sustain active putrescine
and spermidine uptake(15, 16) . On the other hand,
both the putrescine (13) and spermidine preferential (11) carriers in E. coli are ATP-binding cassette
transporters(14) , and the potA subunit of the
spermidine-preferential carrier requires ATP hydrolysis for its
activity(16) . Since the ATPase activity is sufficient to drive
substrate transfer by other bacterial transporters of the same
family(14) , the role of membrane potential in polyamine
transport may be to counteract electrostatic binding of substrates to
the carrier and thus decrease the energy barrier restricting
internalization of these polycations. In this regard, mitochondrial
polyamine transport can be almost exclusively accounted on a non-ohmic,
electrophoretic conductance driven by membrane potential through a
channel-like uniporter(37) . Alternatively, a voltage-dependent
ion conductance could directly participate to the polyamine uptake
mechanism in a countertransport fashion, similar to the
H-ATPase-coupled carriers present in
yeast(28) . The present data on
Rb fluxes would
however appear to rule out a direct role for a K
conductance in polyamine transport. Furthermore, the
insensitivity of
Rb
fluxes to ongoing
polyamine uptake suggests that the latter activity does not
significantly affect membrane potential. The stability of membrane
potential during polyamine uptake might be due to a stoichiometric
counterflow of balancing charges or to the rapid binding of the
internalized substrates to macromolecular
anions(15, 53) .
The inverse relationship noted here between osmolality and polyamine transport agrees with reports on other mammalian cell types(44, 45) , including the mouse mammary gland(43) . Putrescine uptake is also strongly and rapidly increased in response to hyposmotic shock in E. coli(42) , suggesting a general adaptive role for this cellular response to low osmolality. Interestingly, putrescine and spermidine uptake was responsive to osmolality in a more physiologically relevant range here than previously reported(43, 44, 45) . The response observed is clearly triggered by osmotic variations per se and not only by a decrease in ionic strength. Nevertheless, changes in osmolality in vivo are expected to arise most frequently from changes in electrolyte concentrations, and therefore, the dual dependence of polyamine uptake on ionic strength and osmolality could have related regulatory functions. A growing body of evidence indeed suggests that polyamine metabolism and transport are intimately related to the cellular response to osmotic and ionic stress in animal(36, 43, 44, 45, 57) , plants(58) , and bacteria (42) .
The marked pH
dependence of putrescine and spermidine transport observed here had not
been previously reported in mammalian cells. Interestingly, the uptake
process exhibits characteristic substrate differences in pH sensitivity
with a higher optimal pH for putrescine than spermidine. This
differential dependence may either argue in favor of the existence of
distinct carriers for putrescine and polyamines or reflect the
different binding characteristics of the respective substrates to a
common transporter. If the latter interpretation is
correct(5) , a greater inhibition of putrescine uptake by
H may result from a more efficient electrostatic
competition, as suggested above for Na
and
cholinium
. However, the possibility that protonation
of a titratable residue is more critical for putrescine than spermidine
for an efficient interaction with the carrier cannot be discarded,
especially if the extra cationic group of spermidine can compete with
free H
.
Finally, the present results provide the
first demonstration that extracellular divalent cations are essential
for putrescine and spermidine uptake activity in mammalian cells.
Previous reports had pointed to an important role for Ca in polyamine transport, either as a negative (34) or
positive modulator(31, 33, 59) , while others
have failed to demonstrate a significant effect of
Ca
(18) . Most available evidence has
emphasized the involvement of [Ca
]
(30, 31) or transmembrane Ca
fluxes (30, 31, 32) in the uptake
process. As also suggested here with BAPTA-AM (Fig. 7B), [Ca
]
can indeed affect the integrity of the polyamine uptake
mechanism, although perturbation of this component had a minor effect
as compared with strategies aimed at reducing
[Ca
]
. Thus, although calmodulin
antagonists with various specificities inhibit putrescine and
spermidine uptake (28, 29, 30, 31) ,
their effect might owe to their interference with signal transduction
mechanisms responsible for the regulation of transporter activity
and/or Ca
homeostasis(31) . Moreover, the
fact that putrescine uptake inhibition by A23187 was increased by
[Ca
]
would suggest that the
ionophore acts by disrupting the membrane potential upon increased
Ca
and/or Mg
influx (27, 60) and that an increase in free
[Ca
]
per se does not stimulate
putrescine uptake.
The present results strongly suggest that the
[Ca]
requirement for polyamine
transport can in fact be equally satisfied with appropriate
concentrations of other divalent cations such as Mg
and Mn
, the latter being the most potent
effector thus far identified. Furthermore, the divalent cation
requirement for polyamine transport is consistent with tight metal
binding either to the carrier itself or to a closely associated,
essential membrane component, as suggested by the following evidence.
First, prior stripping of the cell monolayers with a chelating agent
was necessary to fully abolish polyamine uptake activity upon deletion
of extracellular Ca
and Mg
. Second,
a high affinity type of interaction between these metals and the
carrier complex is involved, as indicated by the low EC
(50 µM) required for transport restoration by
Mn
and Ca
. Third, channel-mediated
transport of these cations was unlikely involved, since putrescine
uptake activation by Ca
and Mg
was
completely resistant to equimolar additions of Sr
or
Ba
, while the latter metals had weak or no ability,
respectively, to sustain transport. Ca
has been found
to stimulate polyamine uptake in carrot protoplasts likely through cell
surface binding, although Mg
was inactive and no
absolute dependence on divalent cations was demonstrated(59) .
Moderate additivity could at best be demonstrated for the
stimulation of putrescine or spermidine uptake by either
Mn, Ca
, or Mg
,
and thus any of these divalent cations might be physiologically
relevant effectors. A most intriguing finding is that Mn
was a far more potent activator of spermidine than putrescine
transport and was a more efficient activator than Ca
and Mg
. Again, the current uncertainty on the
number of carrier species precludes any firm interpretation of this
difference. Nevertheless, an interesting possibility might be that
tight binding of a highly active divalent metal such as Mn
could regulate the activity of a common putrescine/polyamine
carrier and confer relative specificity in substrate recognition and
transport.
The above findings account for the fact that the sole
deletion of [Ca]
in media
containing the standard Mg
concentration (0.41
mM) only partly abolished putrescine uptake activity ( Fig. 7and Fig. 8). The shared ability of Ca
and Mg
to stimulate carrier activity, as well
as their strong binding to the extracellular surface, might in fact
underlie the reported lack of effect of
[Ca
]
on putrescine uptake in
other systems (18, 30) . In marked contrast with the
present report, spermidine accumulation was found to be enhanced by
depletion of [Ca
]
in human
leukemia cells(31) . The reason for this discrepancy is obscure
but may owe to the much longer period (90 min) used for the uptake
assays in leukemia cells(31) , during which significant
feedback inhibition most likely occur(5, 61) .
Interestingly, Davis and co-workers (34) have described N.
crassa mutants with deregulated putrescine uptake activity,
apparently as a result of a defective protein normally responsible for
the repression of putrescine transport through high-affinity
Ca
binding. Since this putative
Ca
-dependent protein is metabolically unstable and
functions as a transport inhibitor(34) , it may bear analogy
with the short-lived protein responsible for the rapid feedback
inhibition of polyamine uptake in mammalian
cells(5, 61) . If a similar protein occurs in
mammalian cells, Ca
depletion could inactivate it and
thus derepress polyamine uptake, resulting in enhanced net polyamine
accumulation despite reduced carrier activity.
The significance of a
tight association between divalent metals and an essential component of
the polyamine carrier complex remains to be determined. The ability of
transition metals such as Zn, Cu
,
Ni
, and Co
to act as partial,
albeit weak agonists of di- and polyamine uptake, as well as their
relative capacity to inhibit Ca
- or
Mg
-stimulated transport, may suggest the involvement
of an ATPase activity(28, 62, 63) . In yeast
vacuolar polyamine transport, which behaves like a
H
-ATPase-driven uptake system(62) ,
Zn
and Cu
were indeed potent
inhibitors of Mg
-dependent polyamine
uptake(28) . However, as in other V-type ATPases(62) ,
Ca
was also an antagonist(28) , unlike in
ZR-75-1 cells. Furthermore, the apparent extracellular location
of the metal-binding site in ZR-75-1 cells would imply that the
putative ATPase is of an unusual, exofacial type. Alternatively,
divalent cations might form high-affinity coordination complexes with
the carrier that could stabilize the proper folding of the native
molecule(64) . Highly charged substrates such as di- and
polyamines are expected to interact with multiple polar groups of the
carrier protein, and a metal chelate might thus form an integral part
of the ligand recognition site. (
)