ARTICLE
Auteur(s) : Hans
Ebel1, Theodor Günther2
1Charité-Universitätsmedizin Berlin, Campus Benjamin
Franklin, Institut für Klinische Physiologie, Berlin, Germany
2Charité-Universitätsmedizin Berlin, Campus Benjamin
Franklin, Institut für Molekularbiologie und Biochemie, Berlin,
Germany
In erythrocytes, several types of Mg2+ efflux have been
characterized by measuring Mg2+ efflux in different
media: Na+/Mg2+ antiport in NaCl medium [for
a review see 1, 2], choline/Mg2+ antiport in choline·Cl
medium [3] and Mg2+ efflux accompanied by Cl-
efflux in sucrose medium [4-6]. Due to the different properties of
Na+/Mg2+ and choline/Mg2+ antiport
they should represent different entities, but to date the molecular
structure of these transporters is not known. Thus, differentiation
between these possibly different forms of Mg2+ efflux
relies on the use of inhibitors.In rat erythrocytes, both the
Na+/Mg2+ antiport [6] as well as the
choline/Mg2+ antiport [3, 6] can be unspecifically
inhibited by amiloride and quinine or quinidine, allowing no
differentiation between these two forms of Mg2+ efflux.
However, in non Mg2+-loaded rat erythrocytes, imipramine
and other tricyclic drugs carrying a tertiary amine side chain
stimulated Na+/Mg2+ antiport and inhibited
choline/ Mg2+ antiport [7], pointing to the transporters
as different molecules. Interestingly, cinchonine as another
quinoline ring containing compound closely related to quinine, also
inhibited the choline/Mg2+ antiport, but had no effect
on the Na+/Mg2+ antiport [3]. Stimulation of
both Na+/Mg2+ and choline/Mg2+
antiport could be induced by trifluoperazine and by fluvoxamine
[7]. The inhibition of the choline/Mg2+ antiport by
tricyclic drugs carrying a tertiary amine side chain was explained
by competition of the choline-like side chain at the choline
binding site of the exchanger. The stimulation of the
Na+/Mg2+ antiport and of the
choline/Mg2+ antiport remained obscure.In this study we
report that mefloquine, a synthetic quinoline derivate stimulated
the choline/Mg2+ antiport without affecting the
Na+/Mg2+ antiport. The structural
requirements for stimulating the choline/Mg2+ antiport
are discussed by comparing the different drugs.
Materials and methods
Materials
Mefloquine (Lariam®),
rac-erythro-α-2-piperidyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol)
was a kind gift from Hoffmann-La Roche AG (Grenzach, Switzerland).
Nembutal® (pentobarbital sodium) was obtained from Abott
(North Chicago, IL, USA). All other chemicals were purchased at the
highest purity available from Merck®, Darmstadt,
Germany. Filtered, de-ionized and virtually Mg2+-free
water with a resistance of 15-18 MΩ/cm was used for the solutions.
Preparation and incubation of red blood cells
The experiments were conducted with non Mg2+-loaded rat
erythrocytes which have been shown to exhibit a significant
Mg2+ efflux via the Na+/Mg2+
antiport [6] and the choline/Mg2+ antiport [3] without
Mg2+ loading. Red cells were prepared as described
earlier [7]. In brief, blood (6-8 ml) was consistently obtained
from only one anesthetized male Sprague-Dawley rat (50 mg/kg
Nembutal® i.p.), weighing 350-450 g. The vena cava
inferior was catheterized with a heparinized syringe. Portions of
the blood were transferred to heparinized tubes, diluted 1:3 – 1:5
with NaCl medium consisting of 150 mmol·l-1 NaCl, 5
mmol·l-1 D-glucose and 10 mmol·l-1
Hepes-Tris, pH 7.4. The cell suspension was centrifuged at 1000 x g
for 10 min at 24 °C. The plasma and the buffy coat containing
the white cells were aspirated and discarded. The sedimented red
cells were washed twice at 24 °C in NaCl medium. Finally, the red
cells were resuspended and incubated with gentle shaking as a 10%
(v/v) suspension in one of the following media, each containing 5
mmol·l-1 D-glucose and 10 mmol·l-1
Hepes-Tris, pH7.4: (a) 150 mmol·l-1 NaCl (NaCl medium),
(b) 150 mmol·l-1 choline·Cl (choline·Cl medium). To
minimize hemolysis, the cells were handled with utmost caution, the
temperature was kept at 24 °C, and centrifugation was carried out
at 1000 x g. Paired experiments were consistently performed.
Mg2+ efflux
At the beginning of incubation and after 120 min, 1 ml
aliquots of the cell suspensions were centrifuged at 1000 x g for
10 min. To determine Mg2+, the supernatant was
diluted with TCA. The final concentration of TCA was 5% (w/v),
containing 0.1% (w/v) La2O3 and 0.16% (v/v)
HCl. Mg2+ was measured in triplicates by means of atomic
absorption spectrometry (Perkin Elmer, 2380). Mg2+
efflux was calculated from the increase in extracellular
Mg2+ concentration during the time interval, and was
related to the original cell volume measured by hematocrit.
Hematocrit was determined by centrifugation at 1500 x g for
10 min. Hemolysis was measured by determining hemoglobin at
557 nm. Mg2+ efflux was corrected for hemolysis. For
this purpose, Mg2+ was extracted from the sedimented
erythrocytes with 5% (v/v) TCA and was measured after appropriate
dilution of the extract with TCA and
La2O3–HCl as described above.
Statistical analysis
Data were expressed as mean values ± S.E., and statistical
significances were determined by Student’s paired and two tailed
t-test. A value of p < 0.05 was considered significant.
Results and discussion
The effect of 100 μmol·l-1 mefloquine on the
Na+/Mg2+ antiport and the
choline/Mg2+ antiport of non Mg2+-loaded and
non malaria-infected rat erythrocytes is plotted in ( figure 1 ). It can be
seen that mefloquine had no effect on the
Na+/Mg2+ antiport but stimulated the
choline/Mg2+ antiport by approximately 28%. Thus,
through structural variants of quinoline derivates, different
effects on the Na+/Mg2+ antiport and the
choline/Mg2+ antiport could be obtained: inhibition of
both transporters by quinine [3, 6], inhibition of only the
choline/Mg2+ antiport by cinchonine [3], stimulation of
only the choline/Mg2+ antiport by mefloquine. This
different behavior of Na+/Mg2+ and
choline/Mg2+ antiport towards quinoline derivates can be
used as an argument for Na+/Mg2+ and
choline/Mg2+ antiport as different molecular entities.
Table 1( Table 1 ) lists the
structure of several quinoline derivates and tricyclic compounds
that differently affect Na+/Mg2+ and
choline/Mg2+ antiports. As concluded earlier, the
inhibition of the choline/Mg2+ antiport is associated
with the choline-like side chain of the tricyclic ring system in
imipramine and other tricyclic compounds causing a competitive
inhibition of the antiporter [7]. We were not able to identify a
structure-function relationship for the inhibition of the
choline/Mg2+ antiport by quinine or the structurally
related cinchonine, nor for the different effect of both drugs on
the Na+/Mg2+ antiport. However, as to the
stimulation of the choline/Mg2+ antiport by
trifluoperazine and by fluvoxamine, as found in a previous study
[7], and by mefloquine as described in this study, the haloalkyl
CF3 group attached to the benzene ring might be
involved. Mefloquine with two CF3 groups caused a
significantly stronger inhibition of the choline/Mg2+
antiport than fluvoxamine with only one CF3 group, when
tested at the same drug concentration. There was also a lower
effect with trifluoperazine with only one CF3 group.
However, to prevent hemolysis, trifluoperazine was tested at a
lower concentration which may be compensated by its higher
lipophilicity and thus by an increased intercalation into the cell
membrane.
The action of the CF3 group(s) can be explained as
follows: fluorine is the most electronegative element. The C-F bond
with a 44% ionic character is the most ionic of the bonds of carbon
with non-metallic elements. The structure of the CF3
group can be described as a resonance hybrid of various structures,
containing a C+ atom and one negative charge resonating
among the fluorine atoms [8]. When the CF3 groups with
these properties are located near the choline/Mg2+
antiporter, the exchange of Mg2+ for choline may be
enhanced. At present, a more detailed explanation cannot be given
because the structure of the choline/Mg2+ antiporter and
the exchange mechanism are not known.
Mefloquine is a commonly used antimalarial drug. For a review
see [9]. Following infection of human erythrocytes by the malaria
parasite Plasmodium falciparum, a NPP of unknown molecular
structure is induced that has functional and pharmacological
characteristics resembling a volume-regulated anion channel
[10-14]. Although the NPP is mainly permeable to anions and neutral
solutes, it also allows the permeation of inorganic cations such as
Rb+, K+ and of the organic cation choline
[10, 15-19]. This new choline uptake route via NPP is different
from the choline transporter in uninfected human erythrocytes
[15].
The mechanism of quinoline-containing antimalarial drugs has not
yet been elucidated. The main action seems to be an inhibition of
hemoglobin digestion and heme sequestration by the parasite (for a
review see [9]). Moreover, in cultured bovine pulmonary artery
endothelial cells, an inhibition of the volume-regulated anion
channel and of Ca2+-activated Cl--currents by
mefloquine have been reported [20].
Obviously, the stimulation of the choline/Mg2+
antiport by mefloquine is not related to its antimalarial action.
This is supported by several arguments. In human erythrocytes
infected with Plasmodium falciparum, mefloquine did not inhibit the
dramatically increased choline uptake via the NPP [19]. In
non-infected rat erythrocytes the stimulation of the
choline/Mg2+ antiport by mefloquine was observed at a
drug concentration of 100 μmoles·l-1. The
IC50 for inhibition of human intraerythrocyte parasite
growth by mefloquine was only 24 nmol·l-1[19], and the
therapeutic plasma concentration was found in the range of 1 to 5
μmol·l-1[21, 22]. It should be noted that it is unknown
whether rat erythrocytes are less sensitive to mefloquine than
human erythrocytes. Furthermore, since choline is a precursor for
phospholipid headgroup synthesis of the parasite, inhibition of
choline uptake rather than stimulation would be needed to inhibit
parasite growth and the production of the tubulovesicular membrane
network in the erythrocyte cytosol by the parasite. It is also
questionable whether the loss of intracellular Mg2+
produced by the stimulation of the choline/Mg2+ antiport
would suffice to inhibit parasite growth. In in vivo experiments,
only severe Mg2+ deficiency protected mice against
infection with plasmodia which invaded mature erythrocytes [23-26]
and in in vitro experiments, the growth of plasmodia was only
retarded by culturing in Mg2+-free incubation medium
[27, 28].
In summary, in rat erythrocytes, the stimulation of the
choline/Mg2+ antiport by the antimalarial drug
mefloquine, and the inhibition of the choline/Mg2+
antiport by cinchonine, reported by us in a previous study [3], can
be used for differentiating the choline/Mg2+ antiport
from the Na+/Mg2+ antiport which is not
affected by proper concentrations of the drugs. This action of
mefloquine on choline/Mg2+ antiport is not related to
its antimalarial effect.
Table 1 Structural requirements for various drugs
affecting Mg2+ efflux in NaCl medium
(Na+/Mg2+ antiport) and Mg2+
efflux in choline·Cl medium (choline/Mg2+ antiport) in
non Mg2+-loaded, non malaria-infected rat erythrocytes.
Data for imipramine, trifluoperazine and fluvoxamine [7], for
quinine and cinchonine [3] were from previous studies by us. Data
for mefloquine were from figure 1 of this study.
Mg2+ transport is expressed as a percentage difference
to the control, with + for stimulation and – for inhibition.
|
|
Percent difference
|
|
Drug
|
Structure
|
NaCl
|
Choline·Cl
|
|
Imipramine
|
|
+32*
|
-39*
|
|
Quinine
|
|
-24*
|
-62*
|
|
Cinchonine
|
|
-1n.s.
|
-46*
|
|
Trifluoperazine
|
|
+12*
|
+18*
|
|
Fluvoxamine
|
|
+12*
|
+16*
|
|
Mefloquine
|
|
-2 n.s
|
+28*
|
Acknowledgments
The skilful and engaged technical assistance of B. Papanis has been
greatly appreciated. We are grateful for the kind gift of
mefloquine provided to us by Hoffmann-LaRoche AG.
References
1 Günther T, Ebel H. Membrane transport of magnesium. In:
Sigel H, Sigel A, eds. Metal Ions in Biological Systems,
vol. 26. New York, Basel: Marcel Dekker, 1990: 215-25.
2 Vormann J, Günther T. Magnesium transport
mechanisms. In: Birch NJ, ed. Magnesium and the Cell. London,
Boston, San Diego, New York, Sydney: Academic Press, 1993:
137-55.
3 Ebel H, Hollstein M, Günther T. Role of the
choline exchanger in Na+-independent Mg2+
efflux from rat erythrocytes. Biochim Biophys Acta 2002; 1559:
135-44.
4 Günther T, Vormann J. Na+-independent
Mg2+ efflux from Mg2+-loaded human
erythrocytes. FEBS Lett 1989; 247: 181-4.
5 Günther T, Vormann J. Characterization of
Na+-independent Mg2+ efflux from
erythrocytes. FEBS Lett 1990; 271: 149-51.
6 Ebel H, Günther T. Characterization of
Mg2+ efflux from rat erythrocytes non-loaded with
Mg2+. Biochim Biophys Acta 1999; 1421: 353-60.
7 Ebel H, Hollstein M, Günther T. Differential
effect of imipramine and related compounds on Mg2+
efflux from rat erythrocytes. Biochim Biophys Acta 2004; 1667:
132-40.
8 Pauling L. In: The Nature of the Chemical Bond, 3rd
edition. New York: Cornell University Press, Ithaca, 1960: 88-97;
102, and 314-6.
9 Palmer KJ, Holliday SM, Brodgen RN. Mefloquine.
A review of its antimalarial activity, pharmacokinetic properties
and therapeutic efficacy. Drugs 1993; 45: 430-75.
10 Kirk K, Horner HA, Elford BC, Ellory JC,
Newbold CI. Transport of diverse substrates into
malaria-infected erythrocytes via a pathway showing functional
characteristics of a chloride channel. J Biol Chem 1994; 269:
3339-47.
11 Desai SA, Bezrukov SM, Zimmerberg J. A
voltage-dependent channel involved in nutrient uptake by red blood
cells infected with the malaria parasite. Nature 2000; 406:
1001-5.
12 Kirk K. Membrane transport in the malaria-infected
erythrocyte. Physiol Rev 2001; 81: 495-537.
13 Egee S, Lapaix F, Decherf G, Staines HM,
Ellory JC, Doerig C, Thomas SL. A stretch-activated
anion channel is up-regulated by the malaria parasite Plasmodium
falciparum. J Physiol 2002; 542: 795-801.
14 Huber SM, Uhlemann AC, Gamper NL,
Duranton C, Kremsner PG, Lang F. Plasmodium
falciparum activates endogenous Cl- channels of human
erythrocytes by membrane oxidation. EMBO J 2002; 21: 22-30.
15 Kirk K, Wong HY, Elford BC, Newbold CI,
Ellory JC. Enhanced choline and Rb+ transport in
human erythrocytes infected with the malaria parasite Plasmodium
falciparum. Biochem J 1991; 278: 521-5.
16 Kirk K, Horner HA. In search of a selective
inhibitor of the induced transport of small solutes in Plasmodium
falciparum-infected erythrocytes: effects of arylaminobenzoates.
Biochem J 1995; 311: 761-8.
17 Kirk K, Horner HA. Novel anion dependence of
induced cation transport in malaria-infected erythrocytes. J Biol
Chem 1995; 270: 24270-5.
18 Staines HM, Rae C, Kirk K. Increased
permeability of the malaria-infected erythrocyte to organic
cations. Biochim Biophys Acta 2000; 1463: 88-98.
19 Staines HM, Dee BC, Shen MR, Ellory JC.
The effect of mefloquine and volume-regulated anion channel
inhibitors on induced transport in Plasmodium falciparum–infected
human red blood cells. Blood Cells Mol Dis 2004; 32: 344-8.
20 Maertens C, Wie L, Droogmans G, Nilius B.
Inhibition of volume-regulated and calcium-activated chloride
channels by the antimalarial mefloquine. J Pharmacol Exp Ther 2000;
295: 29-36.
21 Hellgren U, Angel VH, Bergqvist Y,
Arvidsson A, Forero-Gomez JS, Rombo L. Plasma
concentrations of sulfadoxine-pyrimethamine and of mefloquine
during regular long term malaria prophylaxis. Trans R Soc Trop Med
Hyg 1990; 84: 46-9.
22 Karbwang J, White NJ. Clinical pharmacokinetics of
mefloquine. Clin Pharmacokinet 1990; 19: 264-79.
23 Maurois P, Gueux E, Rayssiguier Y. Protective
effect of severe magnesium deficiency on Plasmodium chabaudi
infection. Magnesium Res 1989; 2: 183-7.
24 Maurois P, Delcourt P, Slomianny C,
Gueux E, Rayssiguier Y. Effect of dietary magnesium on
the susceptibility of mice to infection by protozoan parasites of
the Apicomplexa and Mastigophora phyla. Magnesium Res 1995; 8:
159-67.
25 Maurois P, Gueux E, Rayssiguier Y. Magnesium
deficiency affects malaria susceptibility in mice. J Am Coll Nutr
1993; 12: 21-5.
26 Maurois P, Delcourt P, Gueux E,
Rayssiguier Y. Magnesium deficiency protects against Babesia
hylomysci and mice become resistant to rechallenge with the
parasite regardless of diet fed. Parasitology 1994; 108: 245-8.
27 Maurois P, Rayssiguier Y, Gueux E,
Dei-Case E. In vivo, in vitro inhibitory effect of strong
magnesium deficiency against malarial infections. Magnesium Res
1988; 1: 118; [Abstract].
28 Hess FI, Kilian A, Söllner W,
Nothdurft HD, Pröll S, Löscher T. Plasmodium
falciparum and Plasmodium berghei: effect of magnesium on the
development of parasitemia. Exp Parasitol 1995; 80: 186-93.
|
Printable version
Version PDF
