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.

EXPERIMENTAL PROCEDURES

Materials

Radiometric Determination of Putrescine and Spermidine Uptake

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 NaHPO (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.

Temperature Dependence of Putrescine and Spermidine Uptake

Effect of Putrescine and Spermidine Uptake on Rb Transport

RESULTS

Temperature Dependence of Polyamine Uptake in ZR-75-1 Breast Cancer Cells

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.

Dependence of Polyamine Uptake on Extracellular Na

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 (). NaHPO 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.

Effect of Ionophores and Extracellular K on Polyamine Uptake

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 mMRbCl, 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.

Effect of Osmolality on Putrescine and Spermidine Uptake

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.

Dependence of Putrescine and Spermidine Uptake on pH

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).

Effect of Divalent Cations on Putrescine and Spermidine Uptake

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 MgA, 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.

DISCUSSION

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. ()

FOOTNOTES

*

This work was supported by grants from the Cancer Research Society Inc. and by the Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed.

¶

Supported by Endorecherche Inc.

()

The abbreviations used are: [Na], extracellular Na; BAPTA-AM, bis-(o-aminophenoxy)-ethane-N,N,N`,N`-tetraacetic acid tetra(acetoxymethyl) ester; [Ca], [Ca], intracellular and extracellular free Ca, respectively; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; [K], extracellular K.

()

R. Poulin and A. E. Pegg, manuscript in preparation.

()

C. Zhao and R. Poulin, unpublished results.

()

R. Poulin, K. Torossian, and M. Lessard, unpublished results.

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