Putrescine Transport Research Articles

Characterization of a Diamine Exporter in Chinese Hamster Ovary Cells and Identification of Specific Polyamine Substrates

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.

Characterization of Simian Malarial Parasite (Plasmodium knowlesi)-induced Putrescine Transport in Rhesus Monkey Erythrocytes: A NOVEL PUTRESCINE CONJUGATE ARRESTS IN VITRO GROWTH OF SIMIAN MALARIAL PARASITE (PLASMODIUM KNOWLESI) AND CURES MULTIDRUG RESISTANT MURINE MALARIA (PLASMODIUM YOELII) INFECTION IN VIVO

A stage-dependent increase in the level of putrescine, spermidine, and spermine during intraerythrocytic growth of Plasmodium knowlesi in rhesus monkey erythrocytes was observed. Further, intraerythrocytic P. knowlesi-induced putrescine influx was found in trophozoite stage-infected erythrocytes and process was time- and temperature-dependent and showed saturable kinetics. Characteristics of induced putrescine influx appears in infected erythrocytes to be close to the normal erythrocytes in terms of affinity of putrescine to the putrescine transporter (Km 34.6 +/- 3.8 microM as normal erythrocytes and Km 37.2 +/- 5.2 microM in infected erythrocytes). However, the difference involves the significant increase in the putrescine influx rate after infection (Vmax = 4.21 nmol/min/10(10) normal erythrocytes, compared with 11.6 nmol/min/10(10) infected erythrocytes). Energy dependence, involvement of -SH group, and noninterference by amino acid, spermidine, and spermine in the putrescine influx process clearly demonstrate the presence of a distinct transporter for putrescine in infected erythrocytes. A putrescine conjugate N1,N4-bis(7-chloroquinoline-4-yl)butane-1, 4-diamine (BCBD) was synthesized, which inhibits the putrescine influx in the P. knowlesi infected erythrocytes (Ki of 43.2 microM) as well as in vitro growth of P. knowlesi (IC50 value, 7.64 +/- 0.97 ng/ml BCBD, 10.8 +/- 0.45 ng/ml chloroquine). Addition of exogenous polyamines failed to reverse the inhibitory effect of BCBD in vitro. Administration of BCBD (24 mg/kg body weight, intraperitoneal, twice a day for 4 days) cured the Swiss mice infected with multidrug-resistant infection of Plasmodium yoelii. Therefore, inhibition of putrescine transport in malaria-infected erythrocytes offers a lead in the search of a new class of chemotherapeutic molecules against malaria.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046–1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (<10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice. © 1997 Elsevier Science B.V. All rights reserved.

Polyamine transport systems of Leishmania donovani promastigotes

The following observations are conjointly indicative of the presence of distinct energydependent, saturable and multiple polyamine transport systems in Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis. Spermidine was influxed with as much as seven times higher rate than putrescine, while both spermidine and putrescine transporters exhibited equally high affinity for the respective polyamine. N-Ethylmaleimide arrested the complete functionality of both the transporters which could be restored by reduced glutathione. Putrescine transporter did not recognize spermine but spermidine was recognized to some extent, while spermidine transporter significantly recognized spermine but putrescine was absolutely spared. A few aromatic diamines viz., diaminobiphenyl and the analogs as well as aliphatic diamines viz., cadaverine and agmatine were selectively recognized by the putrescine transporter only. L. donovani promastigotes grown in presence of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, registered marked upregulation of putrescine transport while spermidine transport was only marginally induced. PA transport systems provide the alternative pool of polyamines in L. donovani promastigotes in the absence of an adequate intracellular PA repertoire.

Extracellular calcium stimulates Na +-dependent putrescine uptake in B16 melanoma cells

The regulation of putrescine transport in difluoromethylornithine-treated B16 melanoma cells by extracellular Ca 2+ has been investigated. It was found that physiological concentrations of Ca 2+ were essential for optimum uptake of putrescine and spermidine. Mg 2+, albeit at higher concentrations, also could potentiate polyamine transport. The maximum rate of putrescine uptake increased from 1698 ± 67 pmol/min/mg DNA in the absence of Ca 2+ to 3100 ± 98 pmol/min/mg DNA in the presence of 0.5 mM Ca 2+. There was no change in K m. While Ca 2+ enhanced transport of both putrescine and spermidine it did not affect the uptake of deoxyglucose, thymidine or leucine. Putrescine did not alter Ca 2+ fluxes suggesting that the two cations do not share a common transport system. The effects of Ca 2+ on putrescine uptake appeared to be mediated extracellularly firstly because Ca 2+ did not potentiate putrescine uptake in the presence of A23187 and secondly, because the effects of Ca 2+ were completely inhibited by the lanthanide Tb 3+, which binds to calcium-dependent proteins and does not readily cross biological membranes. Ca 2+ did not affect putrescine transport in the absence of extracellular Na +. Moreover, the rate of putrescine uptake in the absence of Ca 2+ was similar to that in the absence of extracellular Na +. The results from this study indicate that polyamine transport is stimulated by extracellular Ca 2+ and suggest that Ca 2+ is required for activity of the Na +-dependent transporter only. This transporter appears to possess a regulatory binding site for divalent cations.

Excretion and Uptake of Putrescine by the PotE Protein in Escherichia coli

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529-4533). The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.

Putrescine export in Xenopuslaevis oocytes occurs against a concentration gradient: evidence for a non-diffusional export process

Putrescine export was found to occur by a non-diffusional highly regulated process using Xenopus oocytes as a model system of polyamine transport. Untreated oocytes were observed to possess high endogenous intracellular putrescine and spermidine levels with no detectable polyamine interconversion or biosynthesis over the assay intervals. The putrescine uptake process demonstrated a rapid saturation within a 5 min interval. Spermidine demonstrated a relatively larger uptake capacity with only a minimal ability to export. A kinetic analysis of the concentration-dependence of the putrescine and spermidine uptake processes indicated that the putrescine uptake process may possess two concurrent uptake components while spermidine uptake may possess a two-component process with an allosteric regulation. Elevated intracellular putrescine levels were observed to decrease against a 10-fold higher extracellular concentration gradient in a rapid and specific manner. No noticeable changes in the intracellular levels of other polyamines were observed over the same time interval. The uptake and export rates of putrescine transport also showed a concurrent, rapid and cyclical regulation. These findings support a non-diffusional putrescine export process which is highly regulated.

Loss of Intracellular Putrescine Pool-Size Regulation Induces Apoptosis

Synthesis and uptake are two important regulated mechanisms by which eukaryotic cells maintain polyamine levels. The role that loss of synthesis and/or uptake regulation plays in mediating putrescine toxicity was investigated by comparing toxicity in an ornithine decarboxylase (ODC)-deficient Chinese hamster ovary cell line (C55.7) with a functional putrescine transport system and an ODC-overproducing rat hepatoma cell line (DH23b), which are transport regulation deficient. When C55.7 cells were transfected with either mouse ODC (M) or trypanosome ODC (Tb), intracellular putrescine content increased slightly in C55.7(Tb-ODC), compared to C55.7(M-ODC), due to the lack of response of Tb-ODC to polyamine regulation. The increase in putrescine content resulting from loss of ODC regulation had no impact on cell growth and viability. When the feedback repression of polyamine uptake was blocked with cycloheximide, C55.7 cells transfected with either ODC construct accumulated very high levels of putrescine from the medium, and underwent apoptosis in a putrescine dose-dependent manner. A similar correlation of deregulated putrescine uptake and increased apoptotic cells was observed in DH23b cells. These data demonstrate that loss of feedback regulation on the polyamine transport system, but not ODC activity, is sufficient to induce apoptosis. Thus, downregulation of the transport system is necessary to prevent accumulation of cytotoxic putrescine levels in rodent cells.

2,2′-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5. 0 +/- 0.7, 80 +/- 31, and 16 +/- 3 microM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 +/- 0.5 and 2.7 +/- 1.1 microM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 microM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 microM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.

Effects of cationic diamidines on polyamine content and uptake on Leishmania infantum in in vitro cultures

The effect of a series of cationic diamidines recently synthesized by Ciba Geigy, bearing diarylic (CGP040215A and CGP039937A) or monoarylic moieties (CGP033829A, CGP035537A and CGP036958A), was analyzed on some metabolic targets and cell proliferation of in vitro cultures of Leishmania infantum promastigotes (insect form). The action of these compounds on intracellular polyamine pools and putrescine transport suggests that diarylic structures were more effective than their monoarylic counterparts in depleting polyamine levels and inhibiting putrescine transport, although these processes correlate poorly with the antiproliferative rate of these compounds. Finally, the displacement of cationic diamidines to kDNA observed in the presence of several concentrations of spermidine suggests a possible combined mode of action of these molecules, first depleting intracellular polyamine pools and, then, displacing spermidine from its site of interaction to kDNA.

Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens.

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells.

Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027].

Putrescine active uptake system in the Trypanosomatid Crithidia fasciculata.

Using the insect Trypanosomatid Crithidia fasciculata as a model parasite of mammalian pathogenic flagellates, i.e. Leishmania and Trypanosoma spp., we have studied the kinetic and regulatory characteristics of the polyamine uptake system. Putrescine transport was age-dependent with maximum expression values at the proliferative logarithmic phase. Putrescine transport in Crithidia fasciculata was energy-dependent and against a putrescine concentration gradient. The integrity of the membrane sulfhydryl groups was absolutely required for optimum transport rates. The specificity of this mechanism was studied in the presence of a series of different chain length aliphatic diamines, showing the high specificity for putrescine and the poor effect of this series at the highest concentration analyzed as well as the higher polyamines spermidine and spermine. Finally, the well-known inhibitor of polyamine biosynthesis, DFMO, led to an upward regulation of putrescine uptake correlating with the depletion of intracellular polyamine pool. In addition, the presence of high concentrations of putrescine in the culture medium produced a downward regulation of this system.

Polyamine transport inEscherichia coli

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) inEscherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were periplasmic transport systems consisting of four kinds of proteins: pPT104 clone encoded potA, -B,-C, and -D proteins and pPT79 clone encoded potF, -G, -H, and -I proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones. PotD and -F proteins were periplasmic substrate binding proteins and potA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and potH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of potA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (potE protein) haveing twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine.

Modulation of putrescine transport in rat intestinal brush-border membrane vesicles by fasting and refeeding.

Fasting and refeeding dramatically alter small intestinal mucosal growth which is greatly dependent on polyamine biosynthesis and transport. The aim of this study was therefore to examine the uptake of the diamine putrescine by brush-border membrane vesicles from the small intestine of rats fasted for 3 days or refed a standard diet after a PERIOD OF FASTING. WHILE THE MICHAELIS-MENTEN CONSTANT KM WAS essentially unaltered, the maximum velocity (Vmax) for putrescine uptake was 1.85-fold higher in fasted animals than in ad libitum-fed controls. Refeeding fasted rats for 24 h caused a 31% decrease in the Vmax value that, however, remained 1.27-fold higher than in control RATS, WHILE THE KM VALUE WAS STILL UNCHANGED. FASTING RATS OR refeeding rats after a period of fasting caused only a 13 or 17% increase, respectively, in the value of the constant for the nonsaturable component (P) of putrescine transport relative to the corresponding control condition. Our study also confirms that both the mucosal polyamine biosynthesis and intestinal content are altered by fasting. We suggest that an increased uptake activity may have a conservative role by preventing a substantial loss of tissue polyamines during fasting.

Polyamine transport in Escherichia coli and eukaryotic cells

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) in Escherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were ABC (ATP binding cassette) transporters consisting of four kinds of proteins: pPT104 clone encoded PotA, -B, -C, and -D proteins and pPT79 clone encoded PotF, -G, -H, and I proteins. PotD and -F proteins were periplasmic substrate binding proteins and PotA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and PotH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of PotA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on the membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine. In mouse mammary carcinoma FM3A cells, it was shown that the antizyme, which negatively regulates the amount of ornithine decarboxylase, also negatively regulates the activity of polyamine transport.

Transport and steady-state accumulation of putrescine in brush-border membrane vesicles of rabbit small intestine.

Absorption of polyamines from the lumen is essential for cell proliferation in small intestine but also in other rapidly growing body tissues and tumors. Intestinal uptake of polyamines is thought to involve one or more transport systems, but the characteristics of these systems have not yet been clearly elucidated. Because high levels of putrescine have been identified in intestinal lumen, we explored kinetic, physiochemical, and structural features of uptake of this diamine across rabbit intestinal brush-border membrane vesicles (IBBMV) prepared by 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 of 0.45-145 microM. At both temperatures, kinetics of putrescine transport fitted a model with a single Michaelis-Menten uptake component plus a nonsaturable uptake component. At 37 degrees C, the kinetic parameters for the saturable component of putrescine uptake, Km,app and Vmax,app, were 16.8 +/- 4.7 microM and 19.9 +/- 2.8 pmol.mg protein-1.min-1, respectively. The value of the constant for the nonsaturable component of putrescine uptake (P = 0.45 +/- 0.06 x 10(-8) l.mg protein-1.s-1) suggested this component represented essentially nonspecific binding of putrescine to IBBMV. Cadaverine, spermidine, and spermine were competitive inhibitors of putrescine transport, with inhibition constants equal to 47, 117, and 219 microM, respectively. When effects of a variety of alkyldiamines and structural analogues of polyamines (1 mM) on influx of 5.6 microM putrescine were compared, cadaverine, methylglyoxal bis(guanylhydrazone) (MGBG), and cyclic derivatives of MGBG were found to exhibit the highest inhibitory potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of putrescine transport across the intestinal epithelium: study using isolated brush border and basolateral membrane vesicles of the enterocyte.

Putrescine transport was investigated in isolated brush border and basolateral membrane vesicles prepared from the rabbit enterocyte. Brush border vesicles were oriented right-side-out and basolateral vesicles inside-out, forming a model representing uptake and extrusion across the intestinal epithelium. Putrescine transport across both membranes was initially rapid, and 66% of the equilibrium uptake was achieved within the first minute. According to osmoplots and measurements at 4 degrees C, 20% of total incorporation presented binding to the membrane. In order to estimate actual uptake into the vesicles, Km was calculated from the differences in putrescine incorporation at 37 degrees C and 4 degrees C, and was 12.7 mumol L-1 for brush border uptake and 38.2 mumol L-1 for basolateral extrusion. Putrescine uptake into brush border and basolateral membrane vesicles was not enhanced in the presence of an Na+ gradient. When Na+ was substituted with an uncharged solute, mannitol, putrescine incorporation was increased, indicating that putrescine uptake is not Na(+)-dependent and that cations might interfere with the carrier. Paraquat and methylglyoxalbis(guanylhydrazone), known to share the polyamine transport system, inhibited putrescine incorporation in both membrane vesicle preparations. Basolateral carrier showed significantly higher sensitivity to cations. We conclude that putrescine uptake across the apical membrane and extrusion across the basolateral membrane of the enterocyte are mediated by two different and independent carriers which differ in their electrical properties.

Inorganic Cation Dependence of Putrescine and Spermidine Transport in Human Breast Cancer Cells

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 Ca2+ 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 Ca2+ inhibited putrescine uptake more strongly than chelation of intracellular Ca2+. In fact, bound divalent cations were absolutely required for polyamine transport, as shown after brief chelation of the cell monolayers with EDTA. Either Mn2+, Ca2+, or Mg2+ sustained putrescine uptake activity with high potency (Km = 50-300 microM). Mn2+ was a much stronger activator of spermidine than putrescine uptake, suggesting a specific role for this metal in polyamine transport. Other transition metals (Co2+, Ni2+, Cu2+, and Zn2+) were mixed activators/antagonists of carrier activity, while Sr2+ and Ba2+ were very weak agonists, while not interfering with Ca2+/Mg(2+)-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.

Hormonal and Feedback Regulation of Putrescine and Spermidine Transport in Human Breast Cancer Cells

The properties and regulation of the mammalian polyamine transport system are still poorly understood. In estrogen-responsive ZR-75-1 human breast cancer cells, which display low polyamine biosynthetic activity, putrescine and spermidine were internalized with high affinity (Km = 3.7 and 0.5 microM, respectively) via a single class of saturable transporter shared by both substrate types, or via distinct but closely similar carriers. The Vmax, but not the Km of polyamine transport was rapidly and synergistically up-regulated by estrogens and insulin. The steady decay in transport activity observed in hormone-deprived cells was accelerated by retinoic acid. The enhancement of uptake activity resulting from polyamine depletion was amplified 3-fold by estrogens and insulin despite profound growth inhibition, indicating that the cooperative hormonal induction of polyamine transport is dissociated from cell growth status. Polyamine uptake was under feedback inhibition by at least three distinct mechanisms in these cells, namely (i) the induction of a short-lived protein not actively synthesized without ongoing uptake or upon polyamine deletion; (ii) a more latent, protein synthesis-independent "trans-inhibition" mechanism; and (iii) a post-carrier, cycloheximide-sensitive mechanism limiting substrate accumulation. The complexity of these multiple levels of feedback transport inhibition is in keeping with the cytotoxicity of excessive polyamine content.