[CANCER RESEARCH 42, 1289-1295,
0008-5472/82/0042-OOOOS2.00
April 1982]
Initial Rate Kinetics of the Transport of Adenosine and 4-Amino-7-(/î-Dribofuranosyl)pyrrolo[2,3-d]pyrimidine
(Tubercidin) in Cultured Cells
Eric R. Harley, Alan R. P. Paterson, and Carol E. Cass2
Cancer Research Unit (McEachern Laboratory)
Edmonton, Alberta, Canada T6G 2H7
[E. R. H., C. E. C., A. R. P. P.] and Department
ABSTRACT
A procedure is described for determining early time courses
of nucleoside uptake by cultured cells in suspension. Replicate
samples of cell suspensions were exposed to medium contain
ing 3H-nucleosides for brief intervals (sec) ended by addition
of nitrobenzylthioinosine, a potent inhibitor of nucleoside trans
port that terminated nucleoside uptake virtually instantane
ously. Time courses of nucleoside uptake were constructed
from the cellular content of nucleoside acquired by the replicate
samples during graded intervals of exposure to the labeled
permeant. Such time courses were definitive of cellular uptake
of nucleosides during the first few sec of exposure to permeant
and yielded initial rates of uptake of adenosine and 4-amino-7(/8-o-ribofuranosyl)pyrrolo[2,3-cQpyrimidine
(tubercidin). Defin
ing initial rates of nucleoside uptake as rates of inward trans
port, relationships between transport rates and extracellular
concentrations of these permeants were evaluated in HeLa
cells and in two cultured lines of mouse lymphoma L5178Y
cells that differ in their abilities to phosphorylate adenosine and
tubercidin. Transport rates for these permeants were similar in
the two L5178Y cell types and were saturable in the 3 cell lines
with Km values between 14 and 38 JUM.Adenosine and tuber
cidin were mutually competitive permeants in L5178Y cells,
indicating that they are substrates for the same transport
mechanism.
INTRODUCTION
Under physiological conditions, nucleosides entering most
animal cell types are metabolized rapidly and, after brief inter
vals (sec), are found mainly in the form of nucleotide metabo
lites (15, 23). In many cell types, nucleoside molecules cross
the plasma membrane by way of a nucleoside-specific trans
port mechanism. In the absence of permeant metabolism,
nucleoside transport is a reversible, nonconcentrative process
(1, 4, 13, 17, 24). In cells that metabolize nucleosides, rates of
nucleoside transport may exceed those of subsequent enzy
matic transformation of the permeating molecules, with the
result that time courses of nucleoside uptake are complex (8,
23). Rates of inward transport of nucleosides, defined here as
initial rates of cellular uptake of permeant, may be obtained
directly from definitive time courses of cellular uptake of nu
cleosides. Others have extracted rates of transport from ex
tended time courses of nucleoside uptake by fitting such data
to an integrated rate equation based on a theoretical model of
the transport process (23).
Because nucleoside uptake processes are rapid and their
1 Supported
by the Medical Research Council of Canada and the National
Cancer Institute of Canada.
2 To whom requests for reprints should be addressed.
Received October 27, 1981; accepted January 7, 1982.
APRIL
1982
of Biochemistry
[C. E. C., A. R. P. P.], University of Alberta.
time courses complex, it has been technically difficult to obtain
definitive time courses for cellular uptake of the physiological
nucleosides. However, several current technologies allow as
say of permeant uptake during intervals of a few sec and have
been used to estimate transport rates from time courses: (a)
washing methods to rapidly remove permeant-containing me
dium from attached cells (8); (b) centrifugal pelleting of sus
pended cells under oil to rapidly remove cells from permeantcontaining medium (13, 20, 23); and (c) use of transport
inhibitors to rapidly end intervals of permeant uptake by cells.
The current view of cellular nucleoside transport is that of a
facilitated diffusion process mediated by nucleoside-specific
transport elements of the plasma membrane. Under some
conditions, that process is independent of enzymatic transfor
mations to which influent nucleoside molecules are subject (8,
13, 23). The substrate specificity of this transport mechanism
is broad in that a variety of nucleosides, both physiological and
synthetic, are transported by a single type of mechanism, as
indicated by counter-transport experiments (13, 17, 24) and by
effects on nucleoside permeation of nucleoside transport inhib
itors (16) and of genetic impairment of transporter activity (3,
6,9).
In the present study, we examined the inward transport of an
adenosine analog, tubercidin,3 and of adenosine, and we report
that these permeants have high affinity (Km, 38 /ÕMor less) for
the nucleoside transport mechanism in several types of cul
tured cells. Transport rates were derived from time courses of
cellular uptake of permeant obtained by exposing replicate
samples of suspended cells to 3H-permeant for graded brief
intervals. Intervals of permeant uptake were ended by addition
of the potent inhibitor of nucleoside transport, NBMPR (15,
16), followed at once by centrifugal pelleting of cells under an
oil layer (21). Analysis of rate-concentration
relationships
yielded kinetic characteristics for the transport of adenosine
and tubercidin.
MATERIALS
AND METHODS
Cell Cultures. Stock cultures of HeLa cells were maintained as
described previously (2). To obtain the quantities of cells required for
transport experiments (about 108 cells), inocula from monolayer cul
tures were expanded in spinner cultures and then further expanded in
suspension cultures kept under continuous agitation with a vibratory
mixer (Model E1 Vibro-Mixer; Chemapec, Inc., Hoboken, N. J.). The
medium for the spinner and suspension cultures consisted of calciumfree minimum essential medium supplemented with 5% calf serum,
penicillin (100 units/ml), streptomycin (100 ng/ml), and 2 mw HEPES
(pH 7.4). In suspension cultures, cell concentrations were kept below
3 The abbreviations used are: tubercidin, 4-amino-7-</î-o-ribofuranosyl)pyrrolo[2,3-d]pyrimidine;
NBMPR, 6-[(4-nitrobenzyl)thio-9-/3-D-ribofuranosylpurine; HEPES, 4-<2-hydroxyethyl-1-piperazine-W'-2-ethanesulfonic
acid; MMPR,
6-methylthio-9-/8-o-ribofuranosylpurine.
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E. R. Harley et al.
5 x 105 cells/ml
by dilution, and cell proliferation
was exponential,
with doubling times of about 22 hr.
L5178Y/WT and L5178Y/MMPR
mouse lymphoma cell stocks were
maintained as static cultures in Fisher's medium supplemented with
10% horse serum, penicillin (100 units/ml), streptomycin (100 /¿g/ml),
and 2 mM HEPES (pH 7.4). Cultures were restarted from frozen stocks
at 2-month intervals. To obtain cells for experiments, cultures were
expanded to 800 ml in roller bottles, which were rotated at about 1.5
rpm. Cell concentrations were kept below 5 x 105 cells/ml by dilution,
and doubling times were usually about 11 hr. The L5178Y/MMPR
cell
line was derived from a clone (designated H3) isolated by the soft-agar
technique from adenosine kinase-deficient L5178Y cells selected for
resistance to MMPR by culture in the presence of gradually increasing
concentrations of MMPR," a substrate for adenosine kinase (22).
Chemicals. [2-3H]Adenosine (16 to 18 Ci/mmol) and [G-3H]tubercidin (20 Ci/mmol) were purchased from Moravek Biochemicals, Brea,
Calif.; [canboxy/-'4C]inulin (12.6 mCi/mmol) and [L/-'"C]sucrose (584
mCi/mmol) from Amersham, Oakville, Ontario; and 3H2O from New
England Nuclear (Canada), Montreal, Quebec. Deoxycoformycin was
provided by the Division of Cancer Treatment, National Cancer Institute,
Bethesda, Md.
Nucleoside Uptake. In assays of 3H-permeant uptake, HeLa cells
were suspended in "transport medium" which consisted of NaHCO3free minimum essential medium with 20 mM HEPES (pH 7.4), plus a 12
mM increment in NaCI. The transport medium for L5178Y cells con
sisted of NaHCO3-free Fisher's medium with 20 mM HEPES. Uptake
assays were performed within 30 min after cells were transferred to
transport medium.
Initial rates of nucleoside uptake, here defined as rates of nucleoside
transport, were determined from time courses of cellular uptake of 3Hpermeant measured at 21 -22° except in the experiments of Chart 6.
Time courses of permeant uptake were obtained by incubating replicate
assay mixtures for graded time intervals as described below.
The replicate assay mixtures were prepared in 1.5-ml polypropylene
microcentrifuge tubes (Eppendorf). Added first was 150 ^l of oil (spe
cific gravity, 1.03 g/ml; 84.9 parts Dow Corning 550 silicone oil plus
15.1 parts Fisher 0-119 light paraffin oil) upon which was layered 100
fi\ of transport medium containing 3H-permeant (2 to 5 fiCi/ml). Uptake
intervals were begun by addition of 100 ¡lì
of transport medium con
taining 1 to 2 x 106 HeLa cells or 2 to 6 x 106 L5178Y cells. Intervals
of uptake were ended by addition of 200 ¡i\of transport medium
containing 10 /UMNBMPR, followed immediately by centrifugation for
30 sec (Eppendorf Model 5412 microcentrifuge). For each concentra
tion of 3H-permeant assayed, the 3H that became associated with cells
Data Analysis. The permeant content of cell pellets has been ex
pressed as the "pellet;medium ratio," a term operationally defined as
the ratio of the observed 3H-permeant content of a cell pellet (cpm//il
pellet water) to that of the medium (cpm//il medium). This term is
equivalent to the ratio of the content (moles) of 3H-permeant (and
metabolites thereof) in the cell pellet to the content of 3H-permeant in
a volume of medium equal to the aqueous volume (water space) of the
pellet. In the interpretation of such data, nonconcentrative
cellular
uptake of permeant is indicated when ratios approach unity, and ratios
in excess of unity signify cellular accumulation of permeant and/or
metabolites thereof. With respect to 3H-permeant that became associ
ated with cells during uptake intervals of 0 sec (i.e., the "zero-time"
cellular content of permeant), pellet:medium ratios were 0.1 to 0.2 and
were independent of permeant concentration. Rates of cellular uptake
of permeant in the presence of NBMPR, when expressed in terms of
the pelletrmedium ratio, were also independent of permeant concentra
tion. The units of permeant uptake rates were sec'1 or, after multiplying
by the external concentration, pmol/ftl pellet water/sec.
To extract initial rates from time courses of cellular uptake of 3Hpermeant,
least-squares
parabolas originating
at averaged zero-time
uptake values were fitted to the uptake data for intervals from 0 to 5
sec, and initial rates were obtained from coefficients of the first-order
term in the parabolas (equivalent to theoretical tangents to the curves
at zero-time). Time courses of permeant uptake by cells in the presence
of 10 JIM NBMPR were linear and passed through the averaged zerotime uptake values.
Vmaxand Km values of transport were calculated using an iterative
weighted least-squares algorithm outlined by Cleland (5) for fitting the
nonlinear Michaelis-Menten
rate equation directly. The weights were
proportional to the reciprocals of the variances of the velocities.
Metabolism of Permeant during the Uptake Assay. The metabolism
of 3H-permeant by L5178Y/WT and L5178Y/MMPR
cells was deter
mined under conditions of the uptake assay with the following modifi
cations. The 1.5-ml assay tubes contained 100 fil of 0.56 N perchloric
acid over which was layered 300 jul oil and 200 ¿il
of transport medium
containing 3H-permeant, 3H2O, or['"C]sucrose.
Reactions were started
by addition of 100 fil of transport medium containing 2 to 6 x 106 cells.
Reactions were ended by centrifugation for 30 sec, and tubes were
immediately chilled in ice water. The supernatant and oil layers were
removed as described in the uptake assay, and in each tube, the cell
pellet was resuspended in the perchloric acid extract; the resulting
mixtures were kept at 4°for 20 min. The perchloric acid extraction
during an uptake interval of 0 sec was determined using cells that were
pelleted under oil immediately after exposure to medium containing
both 3H-permeant and NBMPR (final concentration, 5 fiM).
mixtures were adjusted to pH values of 4 to 5 by addition of 200 n\ of
0.5 M tricaprylyl tertiary amine (Alamine 336, Henkel Corp., Kankakee,
III.) in Freon (10), and after centrifugation, the aqueous extracts were
subjected to thin-layer Chromatographie analysis to determine the
distribution of cellular 3H between permeant and metabolites thereof.
Rates of cellular uptake of permeant in assay mixtures completed by
simultaneously dispensing HeLa cell suspension and permeant solution
were the same as uptake rates in assay mixtures completed by se
quential addition of those components. This result indicated that the
latter procedure (the "standard" protocol, described above) provided
Extract samples were applied together with appropriate carriers to thin
layers of silica gel (Eastman Kodak Co., Rochester, NY) and the
chromatograms were developed in 1 M ammonium acetate and 0.01 M
EDTA:90% ethanol (30:70, v/v). In some experiments, extract samples
were also analyzed by 2-dimensional thin-layer chromatography
as
sufficiently rapid mixing. Mixing during the interval of permeant expo
sure did not increase cellular uptake of permeant relative to that found
with the standard protocol.
In determining the 3H content of cell pellets, supernatant fractions of
described by Seipal and Reichert (19). Chromatogram sections were
placed in xylene;detergent scintillant (14) and assayed for 3H content
the assay mixtures were removed by suction and each tube was
washed above the oil layer by addition of 1.5 ml of water, which,
together with most of the oil, was then removed by suction. To solubilize
cell pellets. 0.5 ml of 0.5 M KOH was added to each tube, which was
then capped and incubated at 37°overnight. Tubes with their content
of dissolved cells were uncapped and inverted in plastic counting vials
which contained 8 ml of a xylene:detergent scintillant, and vial contents
were then mixed (14). Twenty-four hr after addition of KOH, samples
were assayed for 3H activity by liquid scintillation counting.
4 C. T. Warnick, H. Muzik, and A. R. P. Paterson, unpublished observations.
1290
by liquid scintillation
counting.
RESULTS
In order to study adenosine transport in cultured cells, it has
been necessary to develop a method capable of resolving time
courses of uptake during the first few sec of exposure of cells
to that permeant. We describe here a method using replicate
assay mixtures in which brief intervals of adenosine uptake by
suspended cells were achieved by the use of a transport
"stopper," NBMPR. The method is applicable to the measure-
CANCER
RESEARCH
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VOL. 42
Initial Rate Kinetics of Adenosine and Tubercidin Transport
ment of nucleoside
transport
in a variety of cell-permeant
ugation through oil measured the total and extracellular water
spaces, respectively. The pellet content of 3H2O or was con
stant after 2 sec of incubation, as was that of [14C]inulin, and
both remained constant thereafter for up to 4 min. The water
space of pellets of L5178Y cells after 30 sec of centrifugation
was 0.72 ±0.07 jil/106 cells, and the [14C]inulin space was
9.6 ± 1.3% of the pellet water space (mean ±S.D. from 28
and 8 determinations in triplicate, respectively, of water and
inulin spaces). These values represent data obtained with wildtype and MMPR-resistant L5178Y cells since the results were
the same for both cell types. In similar experiments, the water
space in pellets of HeLa cells was 1.8 ±0.3 /*l/106 cells and
the inulin space was 19 ±3% of the pellet water space (from
20 and 11 determinations in triplicate, respectively, of water
and inulin spaces).
In pellets of L5178Y/MMPR
cells, water and inulin spaces
and adenosine uptake were directly proportional to the cell
content of assay mixtures over a broad range of cell concen
trations (data not shown). NBMPR-sensitive cellular uptake of
adenosine (0.3 /ÃŒM)
during 10-sec incubations was proportional
to cell concentration, and at the highest tested, 4 x 107 cells/
ml, the substrate concentration was reduced by 15% during
the uptake interval without apparent effect on the proportion
ality between cell number and uptake. The apparent extracel
lular adenosine space, as measured by zero-time pellet content
of adenosine in the presence of the transport inhibitor, NBMPR,
was about twice5 the inulin space and about 20% of the pellet
water space. The basis of the difference between the inulin and
adenosine spaces is not known; however, it is possible that the
larger apparent adenosine space may reflect adsorption to the
exterior cell surface. The extracellular tubercidin space was
also about twice the inulin space in pellets of L5178Y/MMPR
cells.
Use of NBMPR to rapidly stop nucleoside transport is illus
trated in Chart 1. In that experiment, intervals of adenosine
uptake by HeLa cells were ended by (a) centrifugal pelleting of
cells under an oil layer or (b) rapid addition of medium contain
ing NBMPR, followed immediately by pelleting of cells under
an oil layer. Use of the "NBMPR stopper" did not affect slopes
of the time courses of adenosine uptake; however, the ordinate
intercept of time courses was reduced by an amount which
was equivalent to uptake during a 2-sec period. The latter is
evident in the displacement on the abscissa of the points of
intersection of time courses obtained in the presence and
absence of NBMPR which were at -0.08 sec (NBMPR + oil
stop) and -2.1 sec (oil stop alone), respectively. The intersec
tion at zero-time (virtual) of the time courses of adenosine
uptake obtained with (a) cells exposed simultaneously to aden
osine and NBMPR and (b) cells in which adenosine uptake was
stopped by NBMPR indicated that the NBMPR stoppage of
adenosine uptake was essentially instantaneous. Other exper
iments reported here, except those of Chart 6, used the
NBMPR-oil centrifugation procedure to end intervals of cellular
uptake of nucleoside permeants.
The experiment of Chart 2 demonstrated that the progress
5 In 11 determinations with L5178Y/MMPR
cells, the apparent extracellular
adenosine space (± S. D.) was 0.15 ±0.2 jil/106 cells.
APRIL 1982
Oil Stop
0.4
combinations.
The 3H2O and [14C]inulin contents of cell pellets after centrif-
NBMPR Stop
0)
^
0.3
5.
"3.
"5
I
0.2
CO
0>
c
"w
O 0.1
Oil Stop
NBMPR Stop
0 12
Seconds
Chart 1. Methods of stopping permeant uptake. To construct time courses of
adenosine uptake, HeLa cells in replicate assay mixtures were incubated for
graded intervals with 0.1 /IM [2-3H]adenosine. Such intervals were started by
addition of 100-/J portions of cell suspension (1.3 x 10e cells) to 100-p.l portions
of medium containing 0.1 JIM [2-3H]adenosine (760 cpm//il) alone (D, O) or with
16 I/M NBMPR (•.•).Such mixtures were layered over oil in microcentrifuge
tubes. Intervals of adenosine uptake were ended by centrifugation alone ("oil
stop"), or by rapid addition of 200 pi of medium containing 16 pM NBMPR
followed at once by centrifugation ("NBMPR stop"). Points, means of 3 assays;
oars, S.D.
of adenosine uptake by HeLa cells was interrupted at once by
the introduction of NBMPR into the experimental system. Aden
osine uptake by HeLa cells was measured in the absence of
NBMPR, in the presence of NBMPR added at zero time, or
after cells had been exposed to adenosine for 15 sec. Time
courses of cellular uptake of adenosine in the presence of
NBMPR had similar slopes, and their points of intersection with
the time course of cellular uptake in the absence of NBMPR
had abscissa values similar to the times of NBMPR addition.
An NBMPR-insensitive component of uptake was indicated by
the positive slopes of time courses obtained in the presence of
the transport inhibitor.
A similar experiment with L5178Y cells (data not shown) also
indicated virtually instantaneous inhibition by NBMPR of cel
lular uptake of [G-3H]tubercidin (0.2 /¿M).
The fractional inhibi
tion of cellular uptake of tubercidin by 20 JUMNBMPR was the
same whether cells were exposed simultaneously to permeant
and inhibitor or exposed for 5 to 10 min to NBMPR prior to
assay of permeation.
Chart 3 presents time courses for uptake of adenosine or
tubercidin over long time intervals (4 min) by cells that differ in
their ability to phosphorylate these permeants. In L5178Y/WT
cells, the formation of metabolites of tubercidin (Table 1) and
adenosine (Table 2) was the apparent basis for the accumula-
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E. R. Harley et al.
lion of 3H from these permeants to intracellular concentrations
more than 50-fold greater than those in the medium. Permeant
depletion probably caused the time-dependent decline in up
take rates in the experiment of Chart 3 since cells had, after 2
min, incorporated over 40% of the label. Cells of the adenosine
kinase-deficient variant, L5178Y/MMPR, accumulated 3H from
adenosine to concentrations 4.7-fold greater than those in the
medium after 4 min of incubation. Examination of metabolite
formation under conditions similar to those of the uptake assay
(Table 2) indicated that there was little, if any, adenosine kinase
activity in cells of the MMPR-resistant line. Accumulation of 3H
8
&
S '-o
o
o.
from adenosine by the latter cells probably represented metab
olism via inosine since exposure to deoxycoformycin, a potent
inhibitor of adenosine deaminase (25), decreased their uptake
of radioactivity (Table 2). In L5178Y/MMPR cells, concentra
tions of 3H derived from tubercidin were lower than from
CL
D
O
I
0.5
O
C
O)
•o
<
0
60
adenosine, evidently reflecting the fact that metabolism of
tubercidin does not proceed by way of deamination as does
that of adenosine in these adenosine kinase-deficient cells.
Tubercidin is not a substrate for adenosine deaminase (21).
Chart 4 illustrates the means by which initial rates of nucleoside uptake (rates of transport) were extracted from time
120
Seconds
Chart 2. Rapid onset of NBMPR inhibition of adenosine uptake by HeLa cells.
In replicate assay mixtures, HeLa cells (2.0 x IO6/assay) were exposed to 0.1
¡M [3H]adenosine (4700 cpm/pmol) for the indicated intervals (0 to 30 sec)
which were ended by the addition of 200 Ml of medium containing 16 /IM NBMPR
(O), followed at once by pelleting under oil as in Chart 1. With assay mixtures
subjected to uptake intervals of O (G) and 15 (•)sec, centrifugation was delayed
after addition of NBMPR for the times indicated.
Table 1
Formation of metabolites from tubercidin by L5178Y/WTand
L5178Y/MMPR
cells
Presented are the percentages of total intracellular radioactivity present in
metabolites formed after cells were exposed for the times indicated to 1.0 »M[G3H]tubercidin. The procedures used for extraction of metabolites and for Chro
matographie analysis are described in "Materials and Methods." Radioactivity
derived from extracellular tubercidin which accompanied cells during their pel
leting into perchloric acid was computed from measurements of [U-MC]sucrose
and 3H2O in extracts prepared under conditions identical to those used for
measurement of metabolites of tubercidin. This "extracellular"
contribution was
subtracted from the tubercidin
reported below.
content of the extract in obtaining
% of total intracellular
L5178Y/WT
Metabolite
Tubercidin3Tubercidin
5 sec
monophosphate
Tubercidin
diphosphate6Tubercidin
the results
radioactivity
L5178Y/MMPR
3 min
5 sec
3 min
3.623.31.51.39.787.097.60.1
01.296.40.20.21.4
triphosphate56.413.5
Identified using authentic tubercidin as a marker.
" Calculated from 3H activity that cochromatographed
with adenosine phos
phate markers; the identities are presumed.
Table 2
Formation of metabolites from adenosine by L51 78Y/MMPR cells
Presented are the percentages of total intracellular radioactivity present in
metabolites formed after cells were exposed for the times indicated to 1.8 /IM [23H]adenosine. For further details, see legend to Table 1.
1
2
% of total intracellular radioactivity
L5178Y/MMPR
cells
4
Minutes
Chart 3. Long time courses of uptake of adenosine and tubercidin by wildtype and adenosine kinase-deficient cells. Intervals of permeant uptake were
started by adding cells to permeant-containing
medium and were ended by
pelleting cells under oil. L5178Y/WT [adenosine kinase-competent (AK*)] cells
(2.5 x 10Vassay; •,•,A) or L5178Y/MMPR [adenosine kinase-deficient (/\K~)]
secMetaboliteAdenosineAdenineHypoxanthineInosineAMP
5
in
min-dCF2.31.05.07.560.819.82.1+d
cells (3.5 x 10Vassay; D, O, A) were incubated at room temperature with either
0.3 UM [3H]adenosine (2010 cpm//il; •,O. A) or 0.2 JIM [3H]tubercidin (1220
cpm/pl; •D, A) in the presence or absence of 25 fiM NBMPR for the time
intervals indicated. The data represent single assays, except those for uptake in
the presence of NBMPR (A), which represent averaged results from the 4 cellpermeant combinations (standard deviations are within the symbols}.
The water space in the pellets of both cell types was 0.7 ±0.01 fil/106 cells,
and the inulin space was 9 ± 1% of that value. The permeant uptake values
(pellet:medium ratios) shown are ratios of counts in the pellet to counts in a
volume of extracellular medium equal to the water space of the pellet.
1292
ATPIMPGuanine
+ ADP +
+ guanosine0-dCF813.96.017.632.85.022.02.2+dCFi>85.112.60.20.11.8022
a dCF, deoxycoformycin.
" Deoxycoformycin
(1 JIM) was added to cell cultures
uptake experiments.
c There was no detectable radioactivity
15 min before the
in guanine nucleotides.
CANCER
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VOL.
42
Initial Rate Kinetics of Adenosine and Tubercidin Transport
Adenosine
AK* cells
20
-
•-2
ra
OC
E
2
S
8 ?
<D
O
10
l
I
o
"
O
Tubercidin
AK" cells
O 16
o.
64
Nucleoside
128
Concentration
256
(fiM)
'5 <D
o «
oO
1
2345
O
12345
Seconds
a
Chart 4. Initial time course of adenosine and tubercidin uptake by L5178Y/
WT [adenosine kinase-competent (AK*)] and L5178Y/MMPR [adenosine kinasedeficient MK~)] cells. Using replicate assay mixtures, time courses of cellular
0.25
0.50
1 / Nucleoside Concentration
uptake of permeant were determined as in Chart 1. In the assay mixtures, final
concentrations of adenosine (2400 cpm//il) and tubercidin (1500 cpm/jul) were
1 (O), 4 <A), 16 (D), 64 (•),and 256 (•)/UM, and 2 to 4 X 106 cells were
present in each assay. The permeant uptake values (pellet:medium ratios) shown
are ratios of counts in the pellet to counts in a volume of extracellular medium
equal to the water space of the pellet. Each datum represents a single determi
nation, with the exception of the zero-time uptake value (the same in each panel)
which is an average from all of the cell-permeant combinations tested. The lines
are least-squares parabolas constrained to pass through the average zero-time
uptake value for which the standard deviation falls within the symbol.
courses of uptake. Cells were exposed to graded concentra
tions of 3H-labeled adenosine or tubercidin for brief intervals
that were ended by the addition of NBMPR. To obtain values
for zero-time uptake of permeant, cell suspensions were added
to medium containing both 3H-permeant and NBMPR with
immediate
centrifugation.
Such values, expressed
as
pellet:medium ratios, were similar at various permeant concen
trations. The average of 22 measurements of the pellet:medium
ratio was 0.097 ± 0.024. Values for cellular uptake of 3Hpermeant during graded intervals, together with the mean zero-
Chart 5. Kinetics of adenosine and tubercidin uptake by L5178Y/WT [aden
osine kinase-competent OUO] and L5178Y/MMPR [adenosine kinase-deficient
(AK~)] cells. Initial velocities of uptake determined from the time course data of
Chart 4 are plotted against permeant concentration, directly in A (all permeant
uptake rates from Chart 4 are included) or in the reciprocal form in B: adenosine
uptake by L5178Y/WT cells (O); adenosine uptake by L5178Y/MMPR
cells
(•);tubercidin uptake by L5178Y/WT
cells (A); and tubercidin uptake by
L5178Y/MMPR
cells (A). The curved line in A is the least-squares MichaelisMenten solution for all of the Chart 4 rate data combined (Km, 18.8 ±0.7 ¡IM;
Vm,x, 12.5 ± 0.4 pmol/fj/sec).
In 8, rate data from Chart 4 for each cellpermeant combination were treated separately; bars, S.D.
Kinetic characteristics
lineL51
Cell
APRIL 1982
Table 3
of adenosine and tubercidin uptake
(pmol//il/sec)Adenosine
78Y/WTL5178Y/MMPRNucleoside
TubercidinAdenosine 141820
± 2
2"22"V18
18 ± 1
111410
±±
11
Tubercidin21
3019K„±
±
±2"
± 1
14±±1
1
Adenosine
26 ±11
±±
time uptake value, represented time course data to which
parabolas were fitted by computer in order to extract the zerotime velocity ("Materials and Methods"). Thus, the data of
Chart 4 yielded initial velocities of uptake of adenosine and
tubercidin by L5178Y/WT and L5178Y/MMPR
cells, which
are plotted against permeant concentration in Chart 5A. Recip
rocal plots of these data are shown in Chart 56. Similar Kmand
Vmaxvalues of uptake were obtained with both cell lines for
both permeants (Table 3). Because tubercidin was poorly anabolized in the adenosine kinase-deficient cells, the initial rate
kinetics of tubercidin uptake in these cells are evidently those
of inward transport. The similarity of the kinetic characteristics
of permeant uptake in the kinase-competent and kinase-defi
cient cell lines indicates that the initial rate method measured
rates of transport in cells (L5178Y/WT) that metabolized per
meant.
The ability of either adenosine or tubercidin to inhibit the
transport of the other was assessed in the experiments of Chart
6, which used the adenosine kinase-deficient L5178Y/MMPR
1.00
HeLa
a Mean ±S.E.
7 ±3
Estimates for L5178Y cells were obtained from weighted nonlinear leastsquares regression analysis of the data of Charts 4 and 5 and of data (not shown)
from similar experiments with tubercidin, using the algorithm suggested by
Cleland (5) for the Michaelis-Menten equation. Estimates for HeLa cells were
obtained from similar analyses of uptake time courses (data not shown).
cells. Initial rates of cellular uptake of either 3H-permeant, alone
or in the presence of the other (nonisotopic) permeant, were
estimated from the cellular 3H content acquired during the first
2 sec of permeant exposure. Analysis of the influence of
permeant concentration on uptake rates by the Hanes equation
(7) yielded the S/v versus S plots in Chart 6. The parallel form
of the plots indicates that each of the 2 permeants competitively
inhibited the inward transport of the other, suggesting that
adenosine and tubercidin enter L5178Y/MMPR
cells by the
1293
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E. R. Harley et al.
the transporter
tubercidin.
30
cannot
distinguish
between
adenosine
and
DISCUSSION
§20
0)
to
o
CM
O
a
m
10
O
O
16 32
64
128
Tubercidin
256
We have used initial rates of uptake to measure inward fluxes
of adenosine and tubercidin in cells that metabolize these
permeants. Such rates are those of transporter-mediated entry,
the first step in the uptake process. A fundamental requirement
of this approach is the ability to obtain definitive time courses
of permeant uptake, a technological problem of some magni
tude because cellular uptake of some physiological nucleo
sides is so rapid that measurement of uptake during intervals
of less than 5 sec is needed to define the initial aspects of time
courses. Blockade of transporter function by the potent trans
port inhibitor, NBMPR, used in conjunction with centrifugal
pelleting of cells under an oil layer provided a means of achiev
ing such brief intervals of nucleoside uptake. Because early
time courses of uptake of some nucleosides were not linear,
determination of zero-time origins of such time courses was of
critical importance. We have used experimentally determined
origins in the construction of time courses and have estimated
initial rates of nucleoside uptake from such courses by an
objective mathematical procedure.
The inward transport of adenosine measured by these meth
ods was saturable in HeLa cells and in mouse lymphoma
L5178Y cells, and adenosine and tubercidin were mutually
competitive transport substrates in cells of an adenosine kinase-deficient line, L5178Y/MMPR.
Since kinetic analysis of
initial uptake rates gave similar Km and Vma«
values for both
cell-permeant combinations, despite the large differences in
metabolism of tubercidin and adenosine in the 2 lines, we
conclude that the initial uptake rates measured were those of
transport, basically unaffected by permeant metabolism.
128
The apparent affinities for adenosine in the NBMPR-sensitive
256
Adenosine (
nucleoside transport systems in the 3 cell types studied (Km
values, 14 to 38 JUM)are somewhat lower than values reported
Chart 6. Tubercidin (TU) and adenosine (AR): each inhibits the membrane
transport of the other in L5178Y/MMPR
cells. Rates of [G-3H]tubercidin and [2by Lum ef al. (12), who measured adenosine transport in ATP3H]adenosine uptake were estimated from the cellular content of 3H after expo
depleted deoxycoformycin-treated
cultured cells of mouse leu
sure for 2 sec to 3H-permeant under the following conditions. In A, replicate
kemia P388. These investigators reported that adenosine
assay mixtures contained adenosine and tubercidin at the concentrations indi
cated and [G-3HJtubercidin (final concentration, 2340 cpm/fil). In B, replicate
transport is mediated by a single mechanism with Km values of
assay mixtures contained adenosine and tubercidin at the concentrations indi
120 to 160 fiM and Vmaxvalues of 20 to 29 pmol//il pellet
cated and [2-3H]adenosine (final concentration.
5130 cpm/nl). Each datum
water/sec. We have found adenosine transport in untreated
represents the average of triplicate determinations; bars, S.D. The 2-sec intervals
of permeant uptake in this experiment were obtained as in Chart 1 by rapid
cultured P388 cells to have these characteristics: Km, 23 to 34
addition of 200 nl of medium containing 22 (A) or 30 (8),"M NBMPR. In the region
UM; and Vmax,8 to 12 pmol per n\ pellet water per sec (data not
of low substrate concentration, data are presented without symbols. The lines
shown were calculated from parameters obtained from nonlinear weighted leastshown). The higher transporter affinities for adenosine reported
squares regression analysis, as described for the data of Chart 5.
here are probably not due to methodological differences be
tween our studies and those of Lum ef al. (12) because the
same transport mechanism. The K¡value obtained for the
present methods yielded kinetic constants for uridine and
inhibition of tubercidin transport by adenosine (38 ±4 fiM) was
thymidine transport in cultured S49 mouse lymphoma cells
identical to the Km value for tubercidin (36 ±2 fiM). Similarly,
similar to those obtained (18, 23) with the methodology used
the experiment yielded identical K¡and Kmvalues, respectively,
by Lum ef al. (12).
Estimates of extracellular space based on zero-time uptake
for the inhibition of adenosine transport by tubercidin (21.5 ±
of adenosine and tubercidin in NBMPR-stopped experiments
5 ¡J.M)
and for adenosine transport (22 ±2 ¡IM).The apparent
difference between the Km(or K¡)values from Chart 6, A and B, were consistently higher than the inulin space. This difference
was presumably due to variation from one experiment to an
is apparently not attributable to delay in the onset of NBMPR
other, aggravated perhaps by the shortcoming of using an transport inhibitory effects. Others have reported that the inulin
interval rather than a time course to measure velocity. The
space does not account completely for extracellular adenosine
virtual identity of the KÕfor one nucleoside with the Km for the
in pellets of human erythrocytes (11 ).
In summary, we have described a method for measuring
other within each experiment and the similarities of the Kmand
rates of nucleoside transport in suspended cells. The method
of the Vmai<values for the 2 nucleosides (Table 3) suggest that
1294
CANCER
RESEARCH
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VOL. 42
Initial Rate Kinetics of Adenosine and Tubercidin
used a rapid sampling technology to obtain direct measure
ments of initial rates of cellular uptake of nucleoside, which
are, by definition, transport rates. The kinetic characteristics
of adenosine and tubercidin transport were similar in cultured
L5178Y mouse lymphoma cells that either possessed adeno
sine kinase or were deficient in that activity. The nucleoside
transport mechanism in several lines of cultured neoplastic
cells had relatively high affinity (Km, 14 to 38 ¡J.M)
for adenosine.
REFERENCES
1. Cabantchik, Z. I. and Ginsburg, H. Transport of uridine in human red blood
cells. Demonstration of a simple carrier-mediated mechanism. J. Gen. Physiol., 69: 75-96, 1977.
2. Cass, C. E., Dahlig, E., Lau, E. Y., Lynch, T. P., and Paterson, A. R. P.
Fluctuations in nucleoside uptake and binding of the inhibitor of nucleoside
transport, nitrobenzylthioinosine,
during the replication cycle of HeLa cells.
Cancer Res., 39. 1245-1252,
1979.
3. Cass, C. E., Kolassa, N., Uehara, Y., Dahlig-Harley, E., Harley, E. R., and
Paterson, A. R. P. Absence of binding sites for the transport inhibitor
nitrobenzylthioinosine
on nucleoside transport-deficient
mouse lymphoma
cells. Biochim. Biophys. Acta, 649. 769-777, 1981.
4. Cass, C. E., and Paterson, A. R. P. Mediated transport of nucleosides in
human erythrocytes. Accelerative exchange diffusion of uridine and thymidine and specificity toward pyrimidine nucleosides as permeants. J. Biol.
Chem., 247, 3314-3320,
1972.
5. Cleland, W. W. The statistical analysis of enzyme kinetic data. Adv. Enzymol.,
29. 1-32, 1967.
6. Cohen, A., Ullman, B., and Martin, D. W., Jr. Characterization of a mutant
mouse lymphoma cell with deficient transport of purine and pyrimidine
nucleosides. J. Biol. Chem., 254. 112-116, 1979.
7. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics, p. 26. London:
Butterworth, 1979.
8. Heichal, O., Ish-Shalom, D., Koren, R., and Stein, W. D. The kinetic dissec
tion of transport from metabolic trapping during substrate uptake by intact
cells. Uridine uptake by quiescent and serum-activated NIL 8 hamster cells
and their murine sarcoma virus-transformed counterparts. Biochim. Biophys.
Acta, 55?; 169-186, 1978.
9. Jarvis, S. M., and Young, J. D. Nucleoside transport in human and sheep
erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to func
tional nucleoside transport sites. Biochem. J.. 190: 377-383, 1980.
APRIL
1982
Transport
10. Khym, J. X. An analytical system for rapid separation of tissue nucleotides
at low pressures on conventional
ion exchangers.
Clin. Chem., 21:
1245-1252,
1975.
11. Kolassa, N., Plank, B., and Turnheim, K. pH and temperature dependence
of adenosine uptake in human erythrocytes.
Eur. J. Pharmacol., 52.
345-351, 1978.
12. Lum, C. T., März,R., Plagemann, P. G. W., and Wohlhueter, R. M. Adenosine
transport and metabolism in mouse leukemia cells and in canine thymocytes
and peripheral blood leukocytes. J. Cell. Physiol., 101:173-200,
1979.
13. Oliver, J. M., and Paterson, A. R. P. Nucleoside transport. I. A mediated
process in human erythrocytes. Can. J. Biochem., 49. 262-270, 1971.
14. Pande, S. V. Liquid scintillation counting of aqueous samples using Tritoncontaining scintillants. Anal. Biochem., 74. 25-34, 1976.
15. Paterson, A. R. P., Babb, L. R., Paran. J. H., and Cass, C. E. Inhibition by
nitrobenzylthioinosine
of adenosine uptake by asynchronous HeLa cells.
Mol. Pharmacol., 13: 1147-1158,
1977.
16. Paterson, A. R. P., Yang, S., Lau, E. Y., and Cass, C. E. Low specificity of
the nucleoside transport mechanism of RPMI 6410 cells. Mol. Pharmacol.,
16: 900-908, 1979.
17. Plagemann, P. G. W., Marz, R., and Wohlhueter, R. M. Uridine transport in
Novikoff rat hepatoma cells and other cell lines and its relationship to uridine
phosphorylation and phosphorolysis. J. Cell. Physiol., 97. 49-72, 1978.
18. Plagemann, P. G. W., and Wohlhueter. R. M. Permeation of nucleosides,
nucleic acid bases and nucleotides in animal cells. Curr. Top. Membr.
Transp. 14: 225-330, 1980.
19. Seipel, S., and Reichert. U. Two-dimensional thin-layer chromatographyautoradiography of intracellular purine interconversion products. J. Chromatogr. 135: 485-488, 1977.
20. Strauss, P. R.. Sheehan, J. M., and Kashket, E. R. Membrane transport by
murine lymphocytes. J. Exp. Med., Õ44. 1009-1021,
1976.
21. Suhadolnik, R. M. Nucleoside Antibiotics. New York: Wiley Interscience,
1970.
22. Warnick, C. T., and Paterson, A. R. P. Effect of methylthioinosine
on
nucleotide concentrations in L5178Y cells. Cancer Res.. 33. 1711-1715,
1973.
23. Wohlhueter, R. M., Erbe, J., and Plagemann, P. G. W. On the functional
symmetry of nucleoside transport in mammalian cells. Fed. Proc., 39: 1839,
1980.
24. Wohlhueter, R. M., and Plagemann, P. G. W. The roles of transport and
phosphorylation in nutrient uptake in cultured animal cells. Int. Rev. Cytol.,
64: 171-240, 1980.
25. Woo, P. W. K., Dion, H. W., Lange, S. M., Dahl, L. F., and Durham, L. J. A
novel adenosine and ara-A deaminase inhibitor (R)-3-(2-deoxy-^-D-erythropentofuranosyD-3,6,7,8-tetrahydroimidazo[4,5-ol1,3]diazepin-8-ol.
J. Heterocycl. Chem., 11: 641-643, 1964.
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Initial Rate Kinetics of the Transport of Adenosine and
4-Amino-7-( β-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine
(Tubercidin) in cultured Cells
Eric R. Harley, Alan R. P. Paterson and Carol E. Cass
Cancer Res 1982;42:1289-1295.
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