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CELLULAR AND MOLECULAR
Department of Pharmacology, University of Cologne, Cologne, Germany
Received September 16, 2002 ; accepted October 30, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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).
Agmatine is present in plasma and is widely but unevenly distributed in
mammalian tissue (Raasch et al.,
1995).
There is evidence that agmatine acts as an antiproliferative molecule
through induction of the protein antizyme
(Satriano et al., 1998;
Babal et al., 2001). Antizyme
inhibits ornithine decarboxylase and hence polyamine (putrescine, spermine,
and spermidine) biosynthesis. At the same time, antizyme suppresses polyamine
uptake. Concerted intracellular polyamine depletion eventually leads to growth
arrest. Thus, agmatine has been considered a tumor suppressor in the control
of cellular proliferation (Satriano et
al., 1999). In addition, agmatine might serve as a
neurotransmitter or neuromodulator. It is synthesized in specific regions of
the brain, stored in synaptic vesicles, released by depolarization, and
inactivated by agmatinase and diamine oxidase
(Li et al., 1994). Moreover,
agmatine binds to 
2-adrenoceptors and imidazoline binding
sites, and blocks NMDA receptor channels and other ligand-gated cation
channels. It also inhibits nitric oxide synthase and induces release of
peptide hormones. Although the precise function of endogenously released
agmatine in the central nervous system is presently still unclear, there is
obvious therapeutic potential in the treatment of chronic pain (hyperalgesia),
addiction, and brain injury (Reis and
Regunathan, 2000).
Since agmatine carries one or two positive charges at physiological pH, it
will not appreciably cross cellular membranes by simple diffusion. Thus, all
physiological models require the presence of a channel or transporter protein
in the plasma membrane to translocate agmatine. This will allow inactivation
of the transmitter by uptake into metabolizing cells, e.g., in the central
nervous system, or nonexocytotic release from agmatine-producing cells, e.g.,
in the kidney. However, so far, a transport mechanism for agmatine has not
been identified on a molecular level. We have considered the non-neuronal
monoamine transporters EMT, OCT2, and OCT1 (Gründemann et al.,
1994,
1998b;
Okuda et al., 1996) as likely
candidates. In particular, in a recent study we have demonstrated that OCT2
specifically and efficiently transports guanidine, a main component of
agmatine (Gründemann et al.,
1999). The aim of the current study, therefore, was to examine
whether the non-neuronal monoamine transporters accept agmatine as a
substrate. Our results indicate that OCT2 and EMT, but not OCT1, efficiently
translocate agmatine across the plasma membrane and may contribute to both
release and inactivation of this transmitter.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). A cDNA covering the entire open reading frame was
generated by RT-PCR with Pwo Polymerase (Roche Diagnostics, Mannheim,
Germany) and with primers 5'-GAG AGA GAG GAT CCG CCA CCA TGC
CCA CCG TGG ATG ACA TT (forward primer, BamHI site underlined; this
primer introduces a perfect Kozak sequence) and 5'-GAG AGA GAC TCG
AGC TGC AGA GGA TAA CTC CAT CTT (reverse primer, XhoI site
underlined). The amplicon (1.8 kilobases) was cloned, completely sequenced,
and eventually assembled into the BamHI and XhoI sites of
pcDNA3 (Invitrogen, NV Leek, The Netherlands) to yield pcDNA3OCT1h. The amino
acid sequence of our OCT1h clone is identical to the published sequence
(GenBank accession number X98332) except for a single amino acid substitution,
M408V. Sequencing of three independent clones uniformly revealed the same
underlying base disparity, 1294A>G. Total RNA from human kidney (BD Biosciences Clontech, Palo Alto, CA) was used for the cloning of OCT2h. A cDNA covering the entire open reading frame was generated by RT-PCR with LA Taq mixture (Takara Bio, Shiga, Japan) and with primers 5'-GAG AGA GAG GAT CCG CCA CCA TGC CCA CCA CCG TGG ACG AT (forward primer, BamHI site underlined; this primer also introduces a Kozak sequence) and 5'-GAG AGA GAC TCG AGG GCT CAG GGG TAA GTT TGG TT (reverse primer, XhoI site underlined). The amplicon (1.8 kilobases) was cloned, completely sequenced, and eventually assembled into the BamHI and XhoI sites of pcDNA3 to yield pcDNA3OCT2h. The amino acid sequence of our OCT2h clone is identical to the published sequence (GenBank accession number X98333).
Cell Culture and Transfection. The 293 cells (ATCC CRL-1573), a transformed cell line derived from human embryonic kidney, were grown at 37°C in a humidified atmosphere (5% CO2) in plastic culture flasks (Falcon 3112; BD Biosciences, Heidelberg, Germany). The medium was Dulbecco's modified Eagle's medium (31885-023; Invitrogen, Eggenstein, Germany) supplemented with 10% fetal calf serum (Invitrogen). Medium was changed every 2 to 3 days, and the culture was split every 7 days.
The 293 cells were transfected with supercoiled plasmid DNA by lipofection
with the Tfx-50 reagent according to the protocol of the vendor (Promega,
Mannheim, Germany). Stably transfected cells were then selected with geneticin
(G418; Invitrogen) as described
(Gründemann et al., 1997).
Expression of OCT1h and OCT2h was verified by RT-PCR and functional
characterization.
Transport Assays. For measurement of uptake of radiolabeled solutes, cells were grown in surface culture on 60-mm polystyrol dishes (Nunclon 150288; NUNC A/S, Roskilde, Denmark) precoated with 0.1 g/l poly-L-ornithine in 0.15 M boric acid-NaOH, pH 8.4. Cells were used for uptake experiments at a confluence of at least 70%.
Uptake was measured at 37°C. After preincubation for at least 20 min in 4 ml of uptake buffer [125 mM NaCl, 25 mM HEPES-NaOH, pH 7.4, 5.6 mM (+)glucose, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, and 1.2 mM MgSO4], the buffer was replaced with 3 ml of 3H-labeled substrate (at 100 nM if not noted otherwise) in uptake buffer. Incubation was stopped (after 1 min if not noted otherwise) by rinsing the cells four times with each 4 ml of ice-cold uptake buffer. Subsequently, the cells were solubilized with 0.1% (v/v) Triton X-100 in 5 mM Tris-HCl, pH 7.4, and radioactivity was determined by liquid scintillation counting. If not noted otherwise, in this study all inhibitors were absent during preincubation.
Protein Determination. Protein was measured by a modification of the
Bradford method (Zor and Selinger,
1996) with bovine serum albumin as standard.
Calculations and Statistics. Analysis of the time course of
substrate accumulation was based on a one-compartment model as described
earlier (Russ et al., 1992). A
modification for uptake of agmatine into EMTh cells is given in the legend to
Fig. 4. Analysis of saturation
curves and calculation of Ki values have been reported
previously (Schömig et al.,
1993).
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To model the velocity of uptake as a function of pH (see
Fig. 7), for
1-methyl-4-phenylpyridinium iodide (MPP+), a polynomial of the
second degree was used: 
= a0 +
a1 · pH + A2 · pH2.
For agmatine, it follows from S + I = 0.1 µM (S
= concentration of agmatine with a single positive charge; I =
concentration of agmatine with two positive charges), pH =
pKa + log (S/I), and
i = Vmax ·
S/(Km · (1 +
I/Ki) + S) (assuming competitive
inhibition; with I << Ki and S <<
Km, this simplifies to i =
Vmax/Km · S) that
i = Vmax/Km
· 0.1 µM · 10(pH – pKa)/(1 +
10(pH – pKa)). To correct for the p Hdependence of
the transporter as measured by the uptake of MPP+ (see above), the
right half of the last equation was multiplied with (a0 +
a1 · pH + a2 ·
pH2)/(a0 + a1 · 7.5
+ a2 · 7.52). This effects a
normalization relative to pH 7.5, which was chosen arbitrarily. All parameters
were fitted by nonlinear regression.
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Fitted parameters such as Km and Ki values are given as geometric mean with 95% confidence interval. Arithmetic means are given with SEM. p values are from a two-tailed unpaired t test.
Drugs. Radiotracers used were agmatine (H-3, 2220 Bq/pmol; ART-608;
American Radiolabeled Chemicals, St. Louis, MO), 1-methyl-4-phenylpyridinium
iodide (H-3, 2200 Bq/pmol; ART-150; American Radiolabeled Chemicals), and
putrescine (H-3, 2960 Bq/pmol; ART-279, American Radiolabeled Chemicals).
Unlabeled compounds were agmatine sulfate (10144-3; Aldrich Chemical Co.,
Steinheim, Germany), MPP+ (D-048; Sigma/RBI, Natick, MA), and
putrescine (32810; Fluka, Buchs, Switzerland). Disprocynium 24
(1,1'-diisopropyl-2,4-cyanine iodide) was synthesized as described
previously (Russ et al.,
1993). All other chemicals were of analytical grade.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). For
functional expression, the cDNAs were inserted into the eukaryotic expression
vector pcDNA3. The resulting plasmids were used to stably transfect 293 cells,
our standard cell line for heterologous expression of non-neuronal monoamine
transporters. Control cells were stably transfected with empty vector. Stably
transfected cell lines expressing OCT1r, OCT2r, EMTr, and EMTh were available
from previous studies (Breidert et al.,
1998; Gründemann et al.,
1998b,
1999,
2002). To confirm functional expression of OCT1h and OCT2h, saturation of expressed uptake (i.e., total uptake minus total uptake into control cells) of MPP+ was examined (Fig. 1). For OCT1h, the apparent Michaelis-Menten constant, Km, was 32 (95% confidence interval, 27–38) µmol/l and the maximal uptake rate, Vmax, was 1.7 ± 0.1 nmol min–1 mg of protein–1. For OCT2h, the Km was 7.8 (6.2–9.8) µmol/l and Vmax was 0.37 ± 0.01 nmol min–1 mg of protein–1. For both transporters, the Eadie-Hofstee plot is compatible with a single uptake mechanism.
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Characterization of Agmatine Transport. The above-mentioned cell
lines were examined side-by-side in uptake experiments with 0.1 µM
[3H]agmatine as substrate.
Figure 2, upper row, shows
total uptake. To correct for uptake by control cells and for transporter
number, the expressed uptake of agmatine was divided by the expressed uptake
of [3H]MPP+, which was measured in paired assays. The
normalized values (Fig. 2,
lower row) are directly proportional to transport efficiency
(Gründemann et al.,
1999). Agmatine is a good substrate, with a factor of about 0.5
relative to MPP+ uptake, for EMTr and OCT2r, but transport by OCT1r
was about 5-fold lower. An accordant pattern (although on a 4-fold lower level
relative to MPP+ uptake) was observed with the human transporters.
Here OCT1h was about 9-fold less efficient in the transport of agmatine than
EMTh and OCT2h. In contrast to agmatine, uptake of [3H]putrescine
via EMTr, OCT2r, and OCT1r was not significantly different from control
(Fig. 3).
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A detailed analysis of the time course of uptake of 0.1 µM
[3H]agmatine into stably transfected cells expressing EMTh
(Fig. 4) revealed that uptake
was linear with time for at least 15 min, but decreased after about 2 h.
Uptake into control cells was much lower, with rate constants for inwardly
(kin) and outwardly (kout) directed
agmatine fluxes of 1.0 ± 0.1 µl min–1 mg of
protein–1 and 0.027 ± 0.003 min–1,
respectively. To take into account the eventual decline in agmatine content, a
modified fitting function was used (see
Fig. 4 legend). For
EMTh-expressing cells, the initial kin was 6.7 ±
0.3 µl min–1 mg of protein–1 and
kout was 0.012 ± 0.001 min–1. The
maximum uptake amounted to approximately 33 pmol mg of
protein–1. Based on an intracellular water space of 6.7 µl
mg of protein–1 (Martel
et al., 1996) and a conjectured transfection efficiency of 100%, a
50-fold accumulation of agmatine relative to medium can be estimated. Uptake
periods of 1 to 12 min were chosen for subsequent experiments to approximate
initial rates of transport.
Expressed uptake of agmatine via OCT2 or EMT both from rat and human was saturable (Fig. 5a, Table 1), with rather uniform values of Km (1–2 mM) and Vmax (8–16 nmol min–1 mg of protein–1). Because of the low transport activity, the affinity for agmatine of OCT1 was determined by inhibition of MPP+ uptake (Fig. 5b, Table 1). The Ki values for OCT1r and OCT1h were about 10 times higher than the Km values for OCT2 or EMT from the matching species. Thus, compared with OCT1, the affinity of OCT2 and EMT for agmatine is significantly higher.
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A trans-stimulation experiment was made to verify that agmatine is actually transported across the plasma membrane. Cells expressing EMTh were preincubated for 20 min in uptake buffer with 1 mM unlabeled agmatine or MPP+. Control cells were incubated without substrate. After thorough washing, uptake of [3H]MPP+ was measured as usual. As expected, EMTh cells preloaded with MPP+ showed a clear increase in [3H]MPP+ uptake versus control (Fig. 6). On the basis of specific uptake, this acceleration of uptake of extracellular radiolabel by counter-transport of unlabeled intracellular substrate amounts to a factor of 1.9 ± 0.2 (p = 0.0016). With agmatine, stimulation was smaller (factor = 1.4 ± 0.2), yet still significant (p = 0.029). Thus, it is safe to conclude that EMT and, by analogy, OCT2 are carriers of agmatine and not mere binding proteins or channels.
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Since at physiological pH agmatine molecules with one or two positive
charges are present in aqueous solution, we examined which species was the
substrate for the non-neuronal monoamine transporters. Initial rates of uptake
of 0.1 µM [3H]agmatine in buffers adjusted to diverse pH values
were determined with cells expressing EMTh
(Fig. 7a). For controls, uptake
into cells transfected with empty vector and the pH dependence of
MPP+ uptake was recorded in paired assays. Expressed uptake of the
permanent monocation MPP+ increased slightly with increasing pH, as
expected (Schömig et al.,
1992). By contrast, expressed uptake of agmatine was dramatically
dependent on pH. The increase in velocity from pH 7.5 to 8.5 amounted to a
factor of 7.2 ± 0.5. The pKa of the amino group of
agmatine calculated by non-linear regression was 9.07 (8.84–9.44). When
agmatine uptake velocity corrected for pH dependence of the carrier protein
(as measured with MPP+ as substrate) was plotted as a function of
the concentration of singly charged agmatine (as calculated with the
above-mentioned pKa)
(Fig. 7b), a linear relation
was found. In other words, transport velocity was directly proportional to the
concentration of agmatine molecules with a single positive charge. This
indicates that the agmatine species with two positive charges is no substrate
of the non-neuronal monoamine transporters.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Thus, there is a definite need for an integral membrane
protein to remove agmatine from the extracellular space or to release
intracellular agmatine. However, so far, no such transporter has been
identified on a molecular level. In the present study we report that the
non-neuronal monoamine transporters EMT and OCT2, both from rat and human,
efficiently transport agmatine (Fig.
2). By comparison, OCT1 is conspicuously less efficient. The
polyamine and spermine precursor putrescine (1,4-diaminobutane) is
structurally related to agmatine (1-amino-4-guanidobutane), yet there was no
significant transport by any of the (rat) non-neuronal monoamine transporters,
which were highly active for MPP+ transport in paired assays
(Fig. 3). We conclude that OCT2
and EMT specifically accept agmatine as substrate, by virtue of its
guanidinium residue. This, along with the low activity of OCT1, in fact
utterly resembles experiments with histamine as substrate
(Gründemann et al.,
1999).
With Km values of about 1 mM
(Table 1), the affinities of
EMT and OCT2 for agmatine are comparable with other monoamine transmitters
such as noradrenaline, histamine, and 5-hydroxytryptamine
(Breidert et al., 1998;
Gründemann et al.,
1998a,b).
Since Vmax values are relatively high, agmatine transport
is in a low affinity, high turnover mode. The relatively inefficient transport
of agmatine via OCT1 can be fully explained by reduced affinity (measured as
Ki values).
Our exemplary trans-stimulation experiment
(Fig. 6) provides unambiguous
proof that EMTh actually functions as transporter of agmatine, not just as
channel or binding protein. Moreover, the experiment clearly documents that
agmatine transport also works from inside to outside the cell. We expect
similar results for the other non-neuronal monoamine transporters.
Interestingly, in a previous study
(Gründemann et al., 1999)
trans-stimulation of OCT2r by histamine was much stronger (factor =
3.2) than stimulation of EMTh in the present study by agmatine (factor = 1.4),
although both compounds gave similar Vmax values. To
clarify this observation, we have examined whether EMTh transports the
agmatine species with one or two positive charges. The guanidinium group at
one end of agmatine can be considered a permanent cation
(pKa > 13) in aqueous solution. We have not found a
pKa for the amino group at the other end of agmatine in
the literature, but for putrescine the corresponding pKa
is 8.90 at 37°C. A result from the present study
(Fig. 7a) suggests a similar
value for agmatine (pKa = 9.07). Thus, protonation of the
amino group of agmatine depends very much on pH. For example, the fraction of
agmatine molecules with a single positive charge will be 2.6% at pH 7.5 and
21% at pH 8.5. If agmatine2+ was the substrate, then uptake
velocity should decline with increasing pH. However, the opposite was
observed. Agmatine uptake increased dramatically with increasing pH
(Fig. 7a) and was directly
proportional to agmatine+ concentration
(Fig. 7b). Thus, agmatine with
a single positive charge is the sole substrate. This fully explains why
trans-stimulation was lower with agmatine than with histamine: only a
small fraction of intracellular agmatine will carry a single charge; i.e., the
concentration of intracellular substrate is lower than with histamine.
Agmatine2+, after all, could be an inhibitor of transport. However,
since our data for the determination of Km and
Ki (Fig. 5)
do not show any deviation from simple models, we suppose that
agmatine2+ has no significant affinity for the non-neuronal
monoamine transporters. This has further implications. 1) Conversion of the
concentration from total agmatine into agmatine+ indicates that
agmatine+ is actually the best substrate known, for example, for
OCT2r (the transport efficiency relative to MPP+ is 22 and 1.1 for
agmatine+ or guanidine) and EMTh (6.1 and 0.47 for
agmatine+ or histamine)
(Gründemann et al.,
1999). It is unclear why the carriers from rat are more active in
agmatine transport than their human counterparts
(Fig. 2). 2) Based on the same
conversion of concentration, the affinity of OCT2 and EMT for
agmatine+ is quite high: Km = 20–50 µM
(cf. Table 1).
The time course of agmatine uptake into cells expressing EMTh deserves a mention (Fig. 4). After about 2 h, the intracellular agmatine declines. This is unusual and has not been observed in our laboratory with other substrates. The data could be satisfactorily modeled with the variable kin as a function of time (kin = k1 – k2 · t) and the constant kout. The merit of this definition of kin—although it probably makes no sense in terms of a cellular mechanism and a more complex definition may be necessary—is the suggestion that uptake capacity decreases over time. We speculate that agmatine triggers changes that eventually affect the carrier protein. Further work is necessary to clarify this phenomenon.
Uptake of [14C]agmatine was studied previously with synaptosomes
from whole rat brain, at a pH of 7.2
(Sastre et al., 1997). The
transport mechanism was independent of Na+ and had an apparent
Km of 19 mM. Whereas 1 mM noradrenaline or dopamine did
not inhibit uptake of 0.1 mM agmatine significantly, nonspecific
Ca2+ channel blockers Co2+ or Cd2+ at 1 mM or
verapamil at 0.1 mM did so. Noncompetitive inhibition was also achieved by
imidazoline receptor antagonists idazoxan and phentolamine
(Ki = 240 µM for the latter). Sastre et al.
(1997) concluded that agmatine
may be transported through some type of cation channel, especially a
Ca2+ channel. However, from our point of view, the agmatine
transport mechanism in rat brain may well correspond to either OCT2 or EMT for
the following reasons. 1) Affinities may have been underestimated, since the
uptake rates at 10 min were not initial any more. This may account for the
relatively high Km and the lack of effect of noradrenaline
and dopamine. 2) Organic cation transport has been shown to be sensitive to
heavy metal ions (Katsura et al.,
1993). 3) As a large, hydrophobic compound, verapamil likely
blocks all non-neuronal monoamine transporters, as demonstrated for EMTh
(Martel et al., 2001). 4) With
a Ki of 5 µM, phentolamine is a relatively potent
inhibitor, e.g., of EMTh (Gründemann
et al., 2002). 5) Both EMT and OCT2 have been detected in brain
(Gründemann et al., 1997,
1998b;
Busch et al., 1998;
Wu et al., 1998;
Mooslehner and Allen, 1999).
However, the precise localization still has to be established firmly. In the
end, additional work is necessary to resolve whether the agmatine transport
mechanism from rat brain synaptosomes is in fact EMT or OCT2, and whether
distribution of agmatine correlates with transporter expression.
In a recent study with SK-MG-1 cells, an agmatine transport mechanism with
a Km of 8.6 µM and a Vmax of 63
nmol min–1 mg of protein–1 was observed
(Molderings et al., 2001).
Agmatine uptake was independent of Na+ and could be inhibited
neither by corticosterone nor by O-methylisoprenaline, typical
inhibitors of EMT. It was concluded that EMT, among others, is not responsible
for agmatine uptake into SK-MG-1 cells. This is difficult to reconcile with
our data, however, since EMT is functionally expressed in this cell line
(Streich et al., 1996). Thus,
although the available data suggest that in SK-MG-1 cells, a transporter other
than EMT or OCT2 is responsible for agmatine uptake, some additional
verification is desirable. In this respect, the remarkably steep dependence of
agmatine uptake on pH may serve as a distinctive functional marker of the
non-neuronal monoamine transporters. Other recent studies with cultured cells
suggest the existence of a transporter which, in contrast to the non-neuronal
monoamine transporters, accepts putrescine as substrate
(Cabella et al., 2001;
del Valle et al., 2001;
Satriano et al., 2001). The
molecular identity of this carrier is presently unkown.
Molecular identification of transporters of agmatine will help to explore
the versatile actions of this transmitter. For example, it has been shown that
intracellular agmatine, via induction of antizyme, effectively suppresses
synthesis and uptake of polyamines
(Satriano et al., 1998). Since
the resulting depletion of polyamines inhibits cellular proliferation,
agmatine may have a role as tumor suppressor. Interestingly, it has been
suggested that agamatine could be selectively targeted to rapidly
proliferating cells by an increase in membrane transport capacity
(Satriano et al., 1999).
Defining an agmatine transport system is an important aspect of this
hypothesis.
In conclusion, we have identified agmatine transport proteins on a molecular level. OCT2 and EMT both from rat and human efficiently, specifically, and bidirectionally translocate agmatine across the plasma membrane. By contrast, transport of agmatine by the structurally related carrier OCT1 is less efficient. From here on, pharmacological targeting of OCT2 and EMT may help to elucidate the pleiotropic functions of agmatine as a signaling substance. Clearly, a better understanding of agmatine physiology is indispensable for the development of new therapeutic strategies.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: EMT, extraneuronal monoamine transporter; OCT, organic cation transporter; MPP+, 1-methyl-4-phenylpyridinium; RT-PCR, reverse transcriptase polymerase chain reaction; h and r, attached to a protein name, designate species as being human or rat, respectively.
Address correspondence to: D. Gründemann, Department of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Cologne, Germany. E-mail: dirk.gruendemann{at}uni-koeln.de
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