Polyamine uptake in the malaria parasite, Plasmodium falciparum, is dependent on the parasite's membrane potential (original) (raw)
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Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum
International Journal for Parasitology, 2012
Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na + but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential.
Regulation of polyamine transport by polyamines and polyamine analogs
Journal of Cellular Physiology, 1993
Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyarnines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in V , , , , to levels -4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitorinduced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N',N'2-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1 -hr exposure to 10 KM BESPM, V , , , values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400-500 pmol/lO" cells-about 15-20% of the total polyamine pool (-2.8 nmol/1O6 cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 pM produced an increase in SPD and SPM V , , , values, whereas concentrations 3 FM and higher produced a marked suppression of these values. Cells treated with 3 p, M BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15-20%) changes in intracellular polyamine pools. 0 1993 Wiley-Lis5, Inc. T h e polyamines-putrescine (PUT), spermidine (SPD), and spermine (SPM)-found in all mammalian cell types are k n o w n t o b e essential for cell g r o w t h . U n d e r steady-state conditions, i n t r a c e l l u l a r p o l y a m i n e pools are m a i n t a i n e d within a r e l a t i v e l y constant r a n g e by t h e collective effects of a biosynthetic p a t hway, a catabolic pathway, and an u p t a k e mechanism, all o f w h i c h are biologically specific for and sensitively regulated by t h e polyamines themselves (reviewed in Porter e t al., 1992). P o l y a m i n e pools are t y p i c a l l y sustained by a biosynthetic pathway that activates in response to growth-promoting s t i m u l i and down-regulates in t h e presence of exogenous polyamines. T h u s cells can adapt t o t h e availability of exogenous polyamines by u t i l i z i n g t h e m in place o f those t h e y w o u l d 9 1993 WILEY-LISS, INC otherwise synthesize. In cells where t h e biosynthetic pathway i s genetically defective (Pilz e t al., 1990) o r d r u g -i n h i b i t e d ( K r a m e r e t al., 1989), polyamine u p t a k e i s sufficient t o fully sustain cell growth. U n d e r such conditions, cells may up-regulate t h e i r polyamine transport system (Alhonen-Hongisto e t al. , 1980) in ' order t o ensure that i n t r a c e l l u l a r pools a r e maintained.
Transport and metabolism of polyamines in human lymphocytes
International Journal of Biochemistry, 1990
Polyamines are taken up by human peripheral lymphocytes in a concentration, time and pH dependent manner, with an energy-dependent transport system. 2. Each polyamine inhibits the uptake of the others, with the exception of putrescine. Spermine appears to have the highest affinity for the transporter/s. 3. Inhibition by ouabain, amyloride and vanadate suggests that the transport is dependent on Na+. 4. Polyamine content inside the cells increases by ca 6 and 3 times respectively during incubation with spermidine or spermine. 5. The incorporated polyamines are partially transformed into each other.
European Journal of Biochemistry, 1998
Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [ 14 C]putrescine or [ 14 C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (K t ϭ 24.58Ϯ 2.24 µM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (K t ϭ 3.63Ϯ 0.43 µM, K′ t ϭ 0.61Ϯ 0.10 µM). From the K i values, it is inferred that N 1 -5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N 1 -acetylspermine, and then N 1 ,N 12 -diacetylspermine. N 1 -acetylspermidine and N 8acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows:
Polyamine transport systems in the LLC-PK1 renal epithelial established cell line
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1989
LLC-PK I cells were brought to a quiescent state by treatment with DL-2-dif|uoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamlne synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous pntrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependeut and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vma ~ values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent K m = 4.7 -I-0.7pM) than the Na+-independent transporter (apparent K m = 29.8 4-3.5 gM). As a consequence, although the latter transporter had a higher Vm~,, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the anfileukemie agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK t are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.
Polyamines in living organisms
2002
Natural polyamines, putrescine, spermidine and spermine are ubiquitous cell components essential for normal cellular functions and growth. Chemically these compounds are very simple organic aliphatic cations and fully protonated under physiological conditions. There is a strong correlation between proliferation rate of the cells and their polyamine contents. Adjustments of intracellular concentrations of polyamines to physiological requirements are orchestrated by de novo synthesis, polyamine uptake and catabolic reactions. De novo synthesis can in principle be substituted by polyamine uptake from extracellular environment. Over accumulation of polyamines is controlled by release and by a feedback regulation system that involves synthesis of a protein, antizyme that leads to degradation of ornithine decarboxylase and repression of polyamine uptake. The development of specific polyamine biosynthesis inhibitors and structural analogues of polyamines have revealed that maintaining poly...
The Biochemical journal, 1996
Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C,...
Journal of Biological Chemistry
We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent K, values of 0.13 m M for spermine, 0.26 m M for spermidine, and 1 m M for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.
Aliphatic chain length specificity of the polyamine transport system in ascites L1210 leukemia cells
Cancer research, 1984
A series of diamine homologues of putrescine and triamine homologues of spermidine was used to determine the structural specificity of the polyamine transport system in ascites L1210 leukemia cells by measuring their ability to compete with [3H]-putrescine, [3H]spermidine, or [3H]spermine for uptake. Transport specificity among the diamines (as indicated by K1 constants) was greatest for those having chain lengths similar to that of spermidine and least for those similar to putrescine. Among the triamines, transport specificity was greatest for those having an overall chain length similar to those of spermidine and spermine. The homologue competition profiles were relatively the same for [3H]putrescine, [3H]spermidine, or [3H]spermine, suggesting that all three polyamines utilize the same transport system. This was further substantiated by uptake kinetic plots which showed that the three polyamines were competitive inhibitors of one another. In terms of receptor specificity, the ran...