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pH and temperature dependence of adenosine uptake in human erythrocytes
European Journal of Pharmacology, 52 (1978) 345--351
345
© Elsevier/North-Holland Biomedical Press
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE IN HUMAN ERYTHROCYTES NORBERT KOLASSA, BRIGITTE PLANK and KLAUS TURNHEIM
Pharmakologisches Institut, Universit~'t Wien, Wdhringer Strasse 13a, A-1090 Wien, Austria Received 8 May 1978, revised MS received 3 August 1978, accepted 3 August 1978
N. KOLASSA, B. PLANK and K. TURNHEIM, pH and temperature dependence of adenosine uptake in human erythrocytes, European J. Pharmacol. 52 (1978) 345--351. Kinetic analysis of the saturable adenosine uptake in human erythrocytes suggests the existence of two saturable components, distinguished by different Km values (1.4 and 260 pM, respectively, at pH 7.4 and 25°C). Both components were abolished by p-nitrobenzylthioguanosineor dipyridamole. Total uptake was significantly higher at pH 8 than at pH 7 at adenosine concentrations above 2 pM. The increase in uptake at the higher pH was brought about mainly by an increase in the maximum rate of transport of the low-affinity uptake system. With rising temperature the Km and the V of both uptake components increased. No transition temperature was observed between 12 and 37°C. Adenosine uptake
Human erythrocytes
pH dependence
1. Introduction
The coronary vasodilator action of adenosine is short in duration due to the rapid elemination of adenosine from the extracellular space by deamination and by uptake into myocardial and red blood cells (Olsson and Patterson, 1976). An enhancement in the dilator activity of adenosine administered into the coronary artery was observed when the arterial pH was lowered (Raberger et al., 1971). It was the aim of the present investigation to determine whether the reported stronger vasodilator effect of exogenous adenosine in acidosis is brought about by a decrease in cellular adenosine uptake resulting in an elevated adenosine concentration at the extracellularly located putative receptors mediating vasodilation. During the study on the pH dependence of adenosine uptake in human erythrocytes at 25°C, the saturation kinetics at 25°C were found to differ significantly from the results obtained at 0°C (Turnheim et al., 1978): anal-
Temperature dependence
ysis of the data at 25°C revealed two saturable components of adenosine uptake, whereas at 0°C the involvement of only one transport system was apparent. Therefore, the temperature dependence of adenosine uptake in human erythrocytes was also investigated.
2. Materials and methods
The study was carried out using a human erythrocyte suspension as previously described (Turnheim et al., 1978). To obtain the different pH values (7.0--8.0) in the incubation medium (mM: NaC1 125, KC1 4.3, KH2PO4 1.2, MgSO4 1.2, CaC12 2.5, tris(hydroxymethyl)-aminomethane/HC1 buffer 50, glucose 5), the components of the Tris/ HC1 buffer were altered appropriately at the various temperatures (Rauen, 1964). Incubation was initiated by adding 0.5 ml erythrocyte suspension to 1 ml incubation medium containing [8-14C]adenosine and [3H]inulin (Radiochemical Centre, Amersham, U.K.)
346
attaining a final haematocrit of 15%. At the end of the incubation period (usually 2 sec) uptake was stopped by addition of the nucleoside uptake inhibitor p-nitrobenzylthioguanosine (Paterson and Oliver, 1971), NBTGR (generously supplied by Dr. A.R.P. Paterson, University of Alberta, Canada), at a final concentration of 2 pM, followed immediately by centrifugation. Complete and instantenous uptake inhibition by this procedure has been shown previously (Turnheim et al., 1978). Radioactivity was determined in the supernatant and in a perchloric acid extract of the erythrocyte fraction b y means of liquid scintillation counting with appropriate quench correction. The cellular amount of 14C label was calculated from the difference between total ~4C in the erythrocyte fraction and the 14C in the extracellular space (inulin space).
3. Results The time course of the [t4C]adenosine uptake in human erythrocytes was studied in order to estimate the initial rates of uptake for kinetic analysis. 14C-Uptake as a function of time is illustrated in fig. 1 at different concentrations, pH and temperature, in the absence or presence of nucleoside uptake inhibitors. While the cellular amount of 14C increased with time under control conditions, a constant value proportional to the adenosine concentration in the incubation medium was measured in the presence of the inhibitors dipyridamole (Plagemann and Richey, 1974) or N B T G R (Paterson and Oliver, 1971). It is unlikely however that the inhibitor-insensitive uptake represents a diffusional c o m p o n e n t of uptake, since it was independent of time, pH and temperature (fig. 1, table 1). An inulininaccessible space in the immediate vicinity of the erythrocytes, freely accessible to adenosine and not influenced b y dipyridamole or N B T G R may best explain this finding. However, adsorption to the cell mem-
N. KOLASSA ET AL. Adenosine
pH
°C
(pM)
t.o
0.9
o
0.2
74
0
~'
200
8.0
25
~
500
74
37
/ o
0.8
/
0.7
8I
o
0.6
0.5
o
0.4 .E o Z
0.1
.
.
.
.
.
.
Inhibitor
.
Fig. 1. Erythrocyte 14C-concentration divided by the 14C-concentration in the incubation medium, i.e. normalized uptake, as a function of time. Incubation conditions: 0.2 pM [14C]adenosine, pH 7.4, 0°C (o); 200 pM, pH 7.0, 25°C (D); 200 pM, pH 8.0, 25°C (•); 500 gM, pH 7.4, 37°C (A); filled symbols represent the values measured in the presence of 2 pM NBTGR or 4 pM dipyridamole. TABLE 1 14C concentration in the erythrocyte fraction divided by the l a c concentration in the incubation medium containing 2 pM (14C)adenosine and 2 pM NBTGR. pH
Temperature
14Very] 14Cmediurn
(°C) 7.0 7.4 7.6 7.8 8.0
25 25 25 25 25
0.101 0.096 0.100 0.104 0.098
-+ 0.020 1 -+ 0.016 + 0.016 -+ 0.018 -+ 0.022
7.4 7.4 7.4 7.4 7.4
37 31 19 12 0
0.116 0.118 0.109 0.098 0.098
+- 0.024 -+ 0.008 -+ 0.015 +- 0.014 -+ 0.010
1 Mean +_S.D. of 4--8 determinations.
pH AND T E M P E R A T U R E DEPENDENCE OF ADENOSINE UPTAKE
brane or an additional fast transport process, both distinguished by very low affinity and high capacity, cannot be definitely ruled out as other possible explanations (Turnheim et al., 1978). When the total cellular 14C-label was corrected for the amount in the inulin-inaccessible space to give the saturable adenosine uptake, the corrected uptake was linear with time for at least 2 sec indicating that initial rates were measured during this time period *. Therefore, a 2 sec incubation period was chosen for the determination of adenosine uptake in all further experiments. Fig. 2 shows the total adenosine uptake at 25°C as a function of substrate concentration at pH 7--8. Uptake was relatively unaffected by pH variation at low adenosine concentra-
* Linearity of uptake does not necessarily prove unidirectional influx but may also be observed in the presence of backflux of (i) a constant fraction of the amount taken up per time unit or (ii) a fraction proportional to the total amount taken up. The first pos.sibility may be due to intracellular degradation of adenosine to inosine and hypoxanthine with subsequent leakage from the cell; this process would be characterized by varying intercepts on the ordinate of the time dependence of uptake with differences in metabolic fate at different rates of uptake (Schrader et al., Sixma et al., 1976). However, in the present study a c o m m o n intercept on the ordinate was observed under all conditions (fig. 1). The second possibility may be due to degradation of labelled nucleotides to permeable metabolites; this process would be characterized by a reduced slope of the time dependence of uptake in comparison to unidirectional uptake. Leakage of nucleotide metabolites seems to have been negligible in the present study, since in erythrocytes preloaded with [14C]adenosine the cellular 14C~content after addition of NBTGR was constant after prolonged incubation (Turnheim et al., 1978) and NBTGR was shown not to affect hypoxanthine transfer (Patterson and Oliver, 1971). In addition, the fact that the same levels of inosine and hypoxanthine were measured in the supernatant under control conditions and with complete uptake inhibition by NBTGR (Turnheim et al., 1978) argues against detectable leakage of inosine and hypoxanthine. The uptake values in the present study may therefore be considered good estimates for the initial rates of uptake.
347
tions, whereas it was elevated by an increasing pH at high adenosine concentrations (above 2 pM). After the concentration proportional c o m p o n e n t had been subtracted kinetic constants for the saturable adenosine uptake (v) were calculated by least square approximation of parameter estimates (Roos and Pfleger, 1972) assuming t w o saturable components: V = ( V A X S)/(K A + S) + (V B X S)/(KB + S)
where V A and VB are the m a x i m u m velocities, K A and K B the half-saturation constants of carrier-mediated uptake A and B, respectively, and S the substrate concentration. Weighting was performed by dividing the sum of squared deviations by the local variance at each concentration point (Ottaway, 1973). The satisfactory fit of the experimental data by the model comprising two saturable components is illustrated in fig. 3, which is a plot of weighted residuals versus the calculated values (Boxenbaum et al., 1974) at pH 7.6; as is apparent, a roughly equal scatter of weighted residuals around zero was obtained for all values. By contrast, when a model consisting of only a single saturable c o m p o n e n t was employed for parameter approximation, systematic deviations of data from the generated curves were observed (fig. 3). The parallel variations in V A and K A (table 2) with varying pH rule out significant changes of uptake at low adenosine concentrations, whereas the definite increase in VB observed with increasing pH was mainly responsible for the enhancement of uptake at high adenosine concentrations. In addition, adenosine uptake at pH 7.4 was measured at various temperatures. The kinetic constants obtained by use of the calculation procedure given above are presented in table 2. Km and V of both the high- and the low-affinity uptake process showed a consistent tendency to increase with rising temperature. An approximation of parameter estimates to the experimental values obtained at 0°C was only possible for one saturable component, i.e. parameter estimates did n o t converge when a model consisting of t w o
348
N. K O L A S S A ET AL. pH
A
//
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//
)
/ / /
o
/
~
•
78
~.,~,
74
r~
70
76
///"
"3"
10
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2
4
6 pM
B 100
pH
'7
~o
-=
~
~
~
~
8.0
~"~
7.0
d~
100
200
300
400
5 0 0 pM
Fig. 2. [ 1 4 C ] A d e n o s i n e u p t a k e in h u m a n erythrocytes at 25°C as a f u n c t i o n o f c o n c e n t r a t i o n (A: < 6 #M; B: > 2 0 ~ M ) in the i n c u b a t i o n m e d i u m at p H values b e t w e e n 7.0 and 8.0. S y m b o l s represent the m e a n s o f 2 - - 1 0 determinations ( S D a m o u n t e d to a b o u t 10% o f each m e a n value). The h a t c h e d area indicates the N B T G R insensitive c o m p o n e n t o f the total 14C u p t a k e calculated from the m e a n value from table 1 ; the curves were calculated on the basis o f the parameters given in table 2.
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o
.
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8 o -41
0.1
1.0
10
Calculated value (nmoles.m1-1 cell volume .2s -1)
100
Fig. 3. Weighted residuals plotted against calculated values (logarithmic scale). A d e n o s i n e u p t a k e values obtained at pH 7.6 and 2 5 ° C were fitted assuming m o d e l e q u a t i o n s w i t h either one (o) or t w o (o) saturable c o m p o n e n t s .
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE
349
TABLE 2 Apparent kinetic constants of saturable adenosine uptake in human erythrocytes (for calculations see text). pH
Temperature °C
K A (pM)
K B (pM)
VB
VA
(nmoles/ml cell volume/2 sec) 7.0 7.4 7.6 7.8 8.0
25 25 25 25 25
1.00 1.41 1.14 0.81 0.58
_+0.22 1 +- 0.37 _+0.13 _+0.04 _+0.49
80 258 275 180 131
_+ 16 -+ 143 _+ 28 _+ 21 _+ 37
0.83 1.07 0.91 0.55 0.31
_+0.14 -+ 0.28 _+0.14 _+0.01 _+0.22
7.4 7.4 7.4 7.4 7.4 7.4
37 31 25 19 12 0
2.09 1.22 1.41 0.68 0.75
-+ 0.83 -+ 0.35 -+ 0.37 -+ 0.60 _+0.44
1257 359 258 33 14 2.8 _+0.8
-+ 455 -+ 93 -+ 143 -+ 10 _+ 8
2.32 1.46 1.07 0.41 0.19
-+ 0.78 -+ 0.24 -+ 0.28 _+0.34 _+0.10
8_+
1
23_+ 32 -+ 37-+ 44 -+
8 2 1 2
218 60 23 10 3
_+103 _+ 27 + 8 -+ 1 +- 1
0.36 + 0.04
z Mean _+S.D. of 2--4 experiments (determinations in duplicate).
s a t u r a b l e c o m p o n e n t s was used f o r t h e approximation procedure. The Arrhenius plots of the maximum velocities VA and VB o f t h e t w o s a t u r a b l e c o m p o n e n t s o f u p t a k e w e r e linear, indicating the absence of a transition temperature for a d e n o s i n e u p t a k e in h u m a n e r y t h r o c y t e s at
1 2 - - 3 7 ° C (fig. 4). F r o m t h e slopes, an activat i o n e n e r g y o f 16 k c a l / m o l e was derived f o r the high-affinity c o m p o n e n t a n d o f 30 k c a l / mole for the low-affinity component. These values are o f a m a g n i t u d e similar t o t h a t f o u n d in p r e v i o u s investigations o n a d e n o s i n e t r a n s p o r t ( S c h r a d e r et al., 1 9 7 2 ; Berlin, 1973).
1000-
4. Discussion %
~, 100
-
o =
10-
~,
0~
1.0-
o
o ~ ~ , ~
o 0.1-
312
3.'3
a',
3:5 ,o~'°K
Fig. 4. Arrhenius plot of the maximal velocities V A (o) and V B (~',) of the two saturable components of a d e n o s i n e uptake at pH 7.4 in human erythrocytes (table 2).
T h e results suggest t h a t the u p t a k e o f adenosine by human erythrocytes proceeds b y t w o s a t u r a b l e p r o c e s s e s while passive diffusion seems to b e negligible. This d e s c r i p t i o n of adenosine uptake contrasts with previous investigations in w h i c h t h e i n v o l v e m e n t o f o n l y o n e s a t u r a b l e c o m p o n e n t plus passive d i f f u s i o n was r e p o r t e d ( R o o s a n d Pfleger, 1 9 7 2 ; S c h r a d e r et al., 1 9 7 2 ; T a u b e a n d Berlin, 1 9 7 2 ) . In a d d i t i o n t o d i f f e r e n c e s b e t w e e n cell t y p e s and species this d i s c r e p a n c y m a y b e e x p l a i n e d b y difficulties in e s t i m a t i n g corr e c t l y t h e e x t r a c e l l u l a r s p a c e a n d b y testing o n l y relatively l o w s u b s t r a t e c o n c e n t r a t i o n s , t h e r e b y missing t h e l o w - a f f i n i t y u p t a k e . H o w ever, S c h r a d e r et al. ( 1 9 7 2 ) d e s c r i b e d an
350 apparent inhibition of 'adenosine diffusion' by dipyridamole in e r y t h r o c y t e ghosts, which is in agreement with the assumption of a lowaffinity uptake component. Two different transport systems were recently described for the permeation of thymidine in Novikoff rat hepatoma and mouse L cells (Plagemann et al., 1976) as well as for the uptake of adenosine in human platelets (Sixma et al., 1976), murine l y m p h o c y t e s (Strauss et al., 1976) and pig aortic endothelial cells (Pearson et al., 1978). The data obtained for the saturable adenosine uptake at 0°C could not be fitted by the equation comprising two saturable components; only one saturable uptake process was found at this temperature (table 2). A possible explanation may be the insufficient precision of data obtained at this temperature and the similarity of the kinetic constants of the t w o c o m p o n e n t s as extrapolated from the Arrhenius plot (fig. 4) which makes discrimination difficult (Neai, 1972). The Arrhenius plot of the low- and highaffinity adenosine uptake components in human erythrocytes above 12°C showed no transition temperature thus possibly implying that (i) the lipids of the cell membrane underwent no phase transition in the range of temperatures tested, or (ii) the activity of the carrier mechanism was not markedly influenced b y changes in the physical state of the membrane lipids. Also no transition temperature was observed for adenosine uptake in human erythrocyte ghosts (Schrader et al., 1972) and rabbit polymorphonuclear leukocytes (Berlin, 1973). The Km values of adenosine uptake in human erythrocytes decreased with decreasing temperature (table 2). By contrast, the Km values of the saturable adenosine uptake c o m p o n e n t in human erythrocyte ghosts (Schrader et al., 1972) and of the transport of various nucleosides into Novikoff rat hepatoma cells (Plagemann and Erbe, 1975} were found to be not dependent on temperature. However, Berlin (1973) observed a similar change in Km as compared
N. KOLASSA ET AL. to Vmax for adenosine uptake in rabbit alveolar macrophages at various temperatures. This increase in affinity of the putative carrier for adenosine with decreasing temperature may be due to the presumed exothermic nature of the substrate-binding step. There are only few reports on the pH dependence of adenosine uptake in the literature. In general, no marked changes were observed at the various substrate concentrations tested in phosphate buffer (Van Belle, 1969; Sixma et al., 1976). In the present study, using Tris/HC1 buffer, the calculated values of Km and Vmax of the saturable components varied with pH (table 2), b u t no significant differences in overall adenosine uptake by human erythrocytes was found at low adenosine concentrations (<2 ~M) between pH 7 and 8. In spite of this, the uptake varied significantly at high adenosine concentrations (>2 pM) and one may speculate as to whether this pH dependence of adenosine uptake is the reason for the differences in vasodilator efficacy of adenosine at different pH observed in vivo: Raberger et al. (1971) administered 1 pg/kg adenosine into one coronary artery of dogs weighing 30 kg with a basal coronary sinus outflow of about 100 ml/min. In these experiments a dose of 30 pg adenosine (dissolved in 1 ml and injected during 1 sec) yielded a plasma concentration of about 50--100 pM disregarding further dilution. This concentration certaintly lies within the concentration range in which the pH dependence of adenosine uptake in erythrocytes was clearly demonstrated in vitro (fig. 2). Altered rates of elimination of adenosine may therefore be a determining factor for the correlation between the vasodilator action of adenosine and the extracellular pH in vivo.
Acknowledgement The expert technical assistance of Miss G. Brugger is appreciated.
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE
References Berlin, R.D., 1973, Temperature dependence of nucleoside membrane transport in rabbit alveolar macrophages and polymorphonuclear leukocytes, J. Biol. Chem. 248, 4724. Boxenbaum, H.G., S. Riegelman and R.M. Elashoff, 1974, Statistical estimations in pharmacokinetics, J. Pharmacokin. Biopharm. 2,123. Neal, J.L., 1972, Analysis of Michaelis kinetics for two independent, saturable membrane transport functions, J. Theor. Biol. 35, 113. Olsson, R.A. and R.E. Patterson, 1976, Adenosine as a physiological regulator of coronary blood flow, Progr. Mol. Subcell. Biol. 4,227. Ottaway, J.H., 1973, Normalization in the fitting of data by iterative methods, Biochem. J. 134,729. Paterson, A.R.P. and J.M. Oliver, 1971, Nucleoside transport. II. Inhibition by p-nitrobenzylthioguanosine and related compounds, Can. J. Biochem. 49,271. Pearson, J.D., J.S. Carleton, A. Hutchings and J.L. Gordon, 1978, Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture, Biochem. J. 170,265. Plagemann, P.G.W. and D.P. Richey, 1974, Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture, Biochim. Biophys. Acta 344,263. Plagemann, P.G.W. and J. Erbe, 1975, Temperaturedependent changes in activation energies of the transport systems for nucleosides, choline and deoxyglucose of cultured Novikoff rat hepatoma cells and effects of cytochalasin B and lipid solvents, J. Membr. Biol. 25,381. Plagemann, P.G.W., R. Marz and J. Erbe 1976, Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff
351
rat hepatoma and mouse L cells: Evidence for a high Km facilitated diffusion system with wide nucleoside specificity, J. Cell. Physiol. 89, 1. Raberger, G., M. Weissel and O. Kraupp, 1971, The dependence of the effects of i. cor. administered adenosine and of coronary conductance on the arterial pH, pCO2 and buffer capacity in dogs, Naunyn-Schmiedeb. Arch. Pharmakol. 271,301. Rauen, H.M., 1964, Biochemisches Taschenbuch, Vol. 2 (Springer Verlag, Berlin/GSttingen/Heidelber) p. 90. Roos, H. and K. Pfleger, 1972, Kinetics of adenosine uptake by erythrocytes, and the influence of dipyridamole, Mol. Pharmacol. 8,417. Schrader, J., R.M. Berne and R. Rubio, 1972, Uptake and metabolism of adenosine by human erythrocyte ghosts, Amer. J. Physiol. 223,159. Sixma, J.J., J.P.M. Lips, A.M.C. Trieschnigg and H. Holmsen, 1976, Transport and metabolism of adenosine in human blood platelets, Biochim. Biophys. Acta 443, 33. Strauss, P.R., J.M. Sheehan and E.R. Kashket, 1976, Membrane transport by routine lymphocytes. I. A rapid sampling technique as applied to the adenosine and thymidine systems, J. Exptl. Med. 144, 1009. Taube, R.A. and R.D. Berlin, 1972, Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes, Biochim. Biophys. Acta 255, 6. Turnheim, K., B. Plank and N. Kolassa, 1978, Inhibition of adenosine uptake in human erythrocytes by adenosine-5'~arboxamides, xylosyladenine, dipyridamole, hexobendine, and p-nitrobenzylthioguanosine, Biochem. Pharmaeol. (in press). Van Belle, H., 1969, Uptake and deamination of adenosine by blood. Species differences, effect of pH, ions, temperature and metabolic inhibitors, Biochim. Biophys. Acta 192,124.
345
© Elsevier/North-Holland Biomedical Press
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE IN HUMAN ERYTHROCYTES NORBERT KOLASSA, BRIGITTE PLANK and KLAUS TURNHEIM
Pharmakologisches Institut, Universit~'t Wien, Wdhringer Strasse 13a, A-1090 Wien, Austria Received 8 May 1978, revised MS received 3 August 1978, accepted 3 August 1978
N. KOLASSA, B. PLANK and K. TURNHEIM, pH and temperature dependence of adenosine uptake in human erythrocytes, European J. Pharmacol. 52 (1978) 345--351. Kinetic analysis of the saturable adenosine uptake in human erythrocytes suggests the existence of two saturable components, distinguished by different Km values (1.4 and 260 pM, respectively, at pH 7.4 and 25°C). Both components were abolished by p-nitrobenzylthioguanosineor dipyridamole. Total uptake was significantly higher at pH 8 than at pH 7 at adenosine concentrations above 2 pM. The increase in uptake at the higher pH was brought about mainly by an increase in the maximum rate of transport of the low-affinity uptake system. With rising temperature the Km and the V of both uptake components increased. No transition temperature was observed between 12 and 37°C. Adenosine uptake
Human erythrocytes
pH dependence
1. Introduction
The coronary vasodilator action of adenosine is short in duration due to the rapid elemination of adenosine from the extracellular space by deamination and by uptake into myocardial and red blood cells (Olsson and Patterson, 1976). An enhancement in the dilator activity of adenosine administered into the coronary artery was observed when the arterial pH was lowered (Raberger et al., 1971). It was the aim of the present investigation to determine whether the reported stronger vasodilator effect of exogenous adenosine in acidosis is brought about by a decrease in cellular adenosine uptake resulting in an elevated adenosine concentration at the extracellularly located putative receptors mediating vasodilation. During the study on the pH dependence of adenosine uptake in human erythrocytes at 25°C, the saturation kinetics at 25°C were found to differ significantly from the results obtained at 0°C (Turnheim et al., 1978): anal-
Temperature dependence
ysis of the data at 25°C revealed two saturable components of adenosine uptake, whereas at 0°C the involvement of only one transport system was apparent. Therefore, the temperature dependence of adenosine uptake in human erythrocytes was also investigated.
2. Materials and methods
The study was carried out using a human erythrocyte suspension as previously described (Turnheim et al., 1978). To obtain the different pH values (7.0--8.0) in the incubation medium (mM: NaC1 125, KC1 4.3, KH2PO4 1.2, MgSO4 1.2, CaC12 2.5, tris(hydroxymethyl)-aminomethane/HC1 buffer 50, glucose 5), the components of the Tris/ HC1 buffer were altered appropriately at the various temperatures (Rauen, 1964). Incubation was initiated by adding 0.5 ml erythrocyte suspension to 1 ml incubation medium containing [8-14C]adenosine and [3H]inulin (Radiochemical Centre, Amersham, U.K.)
346
attaining a final haematocrit of 15%. At the end of the incubation period (usually 2 sec) uptake was stopped by addition of the nucleoside uptake inhibitor p-nitrobenzylthioguanosine (Paterson and Oliver, 1971), NBTGR (generously supplied by Dr. A.R.P. Paterson, University of Alberta, Canada), at a final concentration of 2 pM, followed immediately by centrifugation. Complete and instantenous uptake inhibition by this procedure has been shown previously (Turnheim et al., 1978). Radioactivity was determined in the supernatant and in a perchloric acid extract of the erythrocyte fraction b y means of liquid scintillation counting with appropriate quench correction. The cellular amount of 14C label was calculated from the difference between total ~4C in the erythrocyte fraction and the 14C in the extracellular space (inulin space).
3. Results The time course of the [t4C]adenosine uptake in human erythrocytes was studied in order to estimate the initial rates of uptake for kinetic analysis. 14C-Uptake as a function of time is illustrated in fig. 1 at different concentrations, pH and temperature, in the absence or presence of nucleoside uptake inhibitors. While the cellular amount of 14C increased with time under control conditions, a constant value proportional to the adenosine concentration in the incubation medium was measured in the presence of the inhibitors dipyridamole (Plagemann and Richey, 1974) or N B T G R (Paterson and Oliver, 1971). It is unlikely however that the inhibitor-insensitive uptake represents a diffusional c o m p o n e n t of uptake, since it was independent of time, pH and temperature (fig. 1, table 1). An inulininaccessible space in the immediate vicinity of the erythrocytes, freely accessible to adenosine and not influenced b y dipyridamole or N B T G R may best explain this finding. However, adsorption to the cell mem-
N. KOLASSA ET AL. Adenosine
pH
°C
(pM)
t.o
0.9
o
0.2
74
0
~'
200
8.0
25
~
500
74
37
/ o
0.8
/
0.7
8I
o
0.6
0.5
o
0.4 .E o Z
0.1
.
.
.
.
.
.
Inhibitor
.
Fig. 1. Erythrocyte 14C-concentration divided by the 14C-concentration in the incubation medium, i.e. normalized uptake, as a function of time. Incubation conditions: 0.2 pM [14C]adenosine, pH 7.4, 0°C (o); 200 pM, pH 7.0, 25°C (D); 200 pM, pH 8.0, 25°C (•); 500 gM, pH 7.4, 37°C (A); filled symbols represent the values measured in the presence of 2 pM NBTGR or 4 pM dipyridamole. TABLE 1 14C concentration in the erythrocyte fraction divided by the l a c concentration in the incubation medium containing 2 pM (14C)adenosine and 2 pM NBTGR. pH
Temperature
14Very] 14Cmediurn
(°C) 7.0 7.4 7.6 7.8 8.0
25 25 25 25 25
0.101 0.096 0.100 0.104 0.098
-+ 0.020 1 -+ 0.016 + 0.016 -+ 0.018 -+ 0.022
7.4 7.4 7.4 7.4 7.4
37 31 19 12 0
0.116 0.118 0.109 0.098 0.098
+- 0.024 -+ 0.008 -+ 0.015 +- 0.014 -+ 0.010
1 Mean +_S.D. of 4--8 determinations.
pH AND T E M P E R A T U R E DEPENDENCE OF ADENOSINE UPTAKE
brane or an additional fast transport process, both distinguished by very low affinity and high capacity, cannot be definitely ruled out as other possible explanations (Turnheim et al., 1978). When the total cellular 14C-label was corrected for the amount in the inulin-inaccessible space to give the saturable adenosine uptake, the corrected uptake was linear with time for at least 2 sec indicating that initial rates were measured during this time period *. Therefore, a 2 sec incubation period was chosen for the determination of adenosine uptake in all further experiments. Fig. 2 shows the total adenosine uptake at 25°C as a function of substrate concentration at pH 7--8. Uptake was relatively unaffected by pH variation at low adenosine concentra-
* Linearity of uptake does not necessarily prove unidirectional influx but may also be observed in the presence of backflux of (i) a constant fraction of the amount taken up per time unit or (ii) a fraction proportional to the total amount taken up. The first pos.sibility may be due to intracellular degradation of adenosine to inosine and hypoxanthine with subsequent leakage from the cell; this process would be characterized by varying intercepts on the ordinate of the time dependence of uptake with differences in metabolic fate at different rates of uptake (Schrader et al., Sixma et al., 1976). However, in the present study a c o m m o n intercept on the ordinate was observed under all conditions (fig. 1). The second possibility may be due to degradation of labelled nucleotides to permeable metabolites; this process would be characterized by a reduced slope of the time dependence of uptake in comparison to unidirectional uptake. Leakage of nucleotide metabolites seems to have been negligible in the present study, since in erythrocytes preloaded with [14C]adenosine the cellular 14C~content after addition of NBTGR was constant after prolonged incubation (Turnheim et al., 1978) and NBTGR was shown not to affect hypoxanthine transfer (Patterson and Oliver, 1971). In addition, the fact that the same levels of inosine and hypoxanthine were measured in the supernatant under control conditions and with complete uptake inhibition by NBTGR (Turnheim et al., 1978) argues against detectable leakage of inosine and hypoxanthine. The uptake values in the present study may therefore be considered good estimates for the initial rates of uptake.
347
tions, whereas it was elevated by an increasing pH at high adenosine concentrations (above 2 pM). After the concentration proportional c o m p o n e n t had been subtracted kinetic constants for the saturable adenosine uptake (v) were calculated by least square approximation of parameter estimates (Roos and Pfleger, 1972) assuming t w o saturable components: V = ( V A X S)/(K A + S) + (V B X S)/(KB + S)
where V A and VB are the m a x i m u m velocities, K A and K B the half-saturation constants of carrier-mediated uptake A and B, respectively, and S the substrate concentration. Weighting was performed by dividing the sum of squared deviations by the local variance at each concentration point (Ottaway, 1973). The satisfactory fit of the experimental data by the model comprising two saturable components is illustrated in fig. 3, which is a plot of weighted residuals versus the calculated values (Boxenbaum et al., 1974) at pH 7.6; as is apparent, a roughly equal scatter of weighted residuals around zero was obtained for all values. By contrast, when a model consisting of only a single saturable c o m p o n e n t was employed for parameter approximation, systematic deviations of data from the generated curves were observed (fig. 3). The parallel variations in V A and K A (table 2) with varying pH rule out significant changes of uptake at low adenosine concentrations, whereas the definite increase in VB observed with increasing pH was mainly responsible for the enhancement of uptake at high adenosine concentrations. In addition, adenosine uptake at pH 7.4 was measured at various temperatures. The kinetic constants obtained by use of the calculation procedure given above are presented in table 2. Km and V of both the high- and the low-affinity uptake process showed a consistent tendency to increase with rising temperature. An approximation of parameter estimates to the experimental values obtained at 0°C was only possible for one saturable component, i.e. parameter estimates did n o t converge when a model consisting of t w o
348
N. K O L A S S A ET AL. pH
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Fig. 2. [ 1 4 C ] A d e n o s i n e u p t a k e in h u m a n erythrocytes at 25°C as a f u n c t i o n o f c o n c e n t r a t i o n (A: < 6 #M; B: > 2 0 ~ M ) in the i n c u b a t i o n m e d i u m at p H values b e t w e e n 7.0 and 8.0. S y m b o l s represent the m e a n s o f 2 - - 1 0 determinations ( S D a m o u n t e d to a b o u t 10% o f each m e a n value). The h a t c h e d area indicates the N B T G R insensitive c o m p o n e n t o f the total 14C u p t a k e calculated from the m e a n value from table 1 ; the curves were calculated on the basis o f the parameters given in table 2.
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Fig. 3. Weighted residuals plotted against calculated values (logarithmic scale). A d e n o s i n e u p t a k e values obtained at pH 7.6 and 2 5 ° C were fitted assuming m o d e l e q u a t i o n s w i t h either one (o) or t w o (o) saturable c o m p o n e n t s .
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE
349
TABLE 2 Apparent kinetic constants of saturable adenosine uptake in human erythrocytes (for calculations see text). pH
Temperature °C
K A (pM)
K B (pM)
VB
VA
(nmoles/ml cell volume/2 sec) 7.0 7.4 7.6 7.8 8.0
25 25 25 25 25
1.00 1.41 1.14 0.81 0.58
_+0.22 1 +- 0.37 _+0.13 _+0.04 _+0.49
80 258 275 180 131
_+ 16 -+ 143 _+ 28 _+ 21 _+ 37
0.83 1.07 0.91 0.55 0.31
_+0.14 -+ 0.28 _+0.14 _+0.01 _+0.22
7.4 7.4 7.4 7.4 7.4 7.4
37 31 25 19 12 0
2.09 1.22 1.41 0.68 0.75
-+ 0.83 -+ 0.35 -+ 0.37 -+ 0.60 _+0.44
1257 359 258 33 14 2.8 _+0.8
-+ 455 -+ 93 -+ 143 -+ 10 _+ 8
2.32 1.46 1.07 0.41 0.19
-+ 0.78 -+ 0.24 -+ 0.28 _+0.34 _+0.10
8_+
1
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8 2 1 2
218 60 23 10 3
_+103 _+ 27 + 8 -+ 1 +- 1
0.36 + 0.04
z Mean _+S.D. of 2--4 experiments (determinations in duplicate).
s a t u r a b l e c o m p o n e n t s was used f o r t h e approximation procedure. The Arrhenius plots of the maximum velocities VA and VB o f t h e t w o s a t u r a b l e c o m p o n e n t s o f u p t a k e w e r e linear, indicating the absence of a transition temperature for a d e n o s i n e u p t a k e in h u m a n e r y t h r o c y t e s at
1 2 - - 3 7 ° C (fig. 4). F r o m t h e slopes, an activat i o n e n e r g y o f 16 k c a l / m o l e was derived f o r the high-affinity c o m p o n e n t a n d o f 30 k c a l / mole for the low-affinity component. These values are o f a m a g n i t u d e similar t o t h a t f o u n d in p r e v i o u s investigations o n a d e n o s i n e t r a n s p o r t ( S c h r a d e r et al., 1 9 7 2 ; Berlin, 1973).
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Fig. 4. Arrhenius plot of the maximal velocities V A (o) and V B (~',) of the two saturable components of a d e n o s i n e uptake at pH 7.4 in human erythrocytes (table 2).
T h e results suggest t h a t the u p t a k e o f adenosine by human erythrocytes proceeds b y t w o s a t u r a b l e p r o c e s s e s while passive diffusion seems to b e negligible. This d e s c r i p t i o n of adenosine uptake contrasts with previous investigations in w h i c h t h e i n v o l v e m e n t o f o n l y o n e s a t u r a b l e c o m p o n e n t plus passive d i f f u s i o n was r e p o r t e d ( R o o s a n d Pfleger, 1 9 7 2 ; S c h r a d e r et al., 1 9 7 2 ; T a u b e a n d Berlin, 1 9 7 2 ) . In a d d i t i o n t o d i f f e r e n c e s b e t w e e n cell t y p e s and species this d i s c r e p a n c y m a y b e e x p l a i n e d b y difficulties in e s t i m a t i n g corr e c t l y t h e e x t r a c e l l u l a r s p a c e a n d b y testing o n l y relatively l o w s u b s t r a t e c o n c e n t r a t i o n s , t h e r e b y missing t h e l o w - a f f i n i t y u p t a k e . H o w ever, S c h r a d e r et al. ( 1 9 7 2 ) d e s c r i b e d an
350 apparent inhibition of 'adenosine diffusion' by dipyridamole in e r y t h r o c y t e ghosts, which is in agreement with the assumption of a lowaffinity uptake component. Two different transport systems were recently described for the permeation of thymidine in Novikoff rat hepatoma and mouse L cells (Plagemann et al., 1976) as well as for the uptake of adenosine in human platelets (Sixma et al., 1976), murine l y m p h o c y t e s (Strauss et al., 1976) and pig aortic endothelial cells (Pearson et al., 1978). The data obtained for the saturable adenosine uptake at 0°C could not be fitted by the equation comprising two saturable components; only one saturable uptake process was found at this temperature (table 2). A possible explanation may be the insufficient precision of data obtained at this temperature and the similarity of the kinetic constants of the t w o c o m p o n e n t s as extrapolated from the Arrhenius plot (fig. 4) which makes discrimination difficult (Neai, 1972). The Arrhenius plot of the low- and highaffinity adenosine uptake components in human erythrocytes above 12°C showed no transition temperature thus possibly implying that (i) the lipids of the cell membrane underwent no phase transition in the range of temperatures tested, or (ii) the activity of the carrier mechanism was not markedly influenced b y changes in the physical state of the membrane lipids. Also no transition temperature was observed for adenosine uptake in human erythrocyte ghosts (Schrader et al., 1972) and rabbit polymorphonuclear leukocytes (Berlin, 1973). The Km values of adenosine uptake in human erythrocytes decreased with decreasing temperature (table 2). By contrast, the Km values of the saturable adenosine uptake c o m p o n e n t in human erythrocyte ghosts (Schrader et al., 1972) and of the transport of various nucleosides into Novikoff rat hepatoma cells (Plagemann and Erbe, 1975} were found to be not dependent on temperature. However, Berlin (1973) observed a similar change in Km as compared
N. KOLASSA ET AL. to Vmax for adenosine uptake in rabbit alveolar macrophages at various temperatures. This increase in affinity of the putative carrier for adenosine with decreasing temperature may be due to the presumed exothermic nature of the substrate-binding step. There are only few reports on the pH dependence of adenosine uptake in the literature. In general, no marked changes were observed at the various substrate concentrations tested in phosphate buffer (Van Belle, 1969; Sixma et al., 1976). In the present study, using Tris/HC1 buffer, the calculated values of Km and Vmax of the saturable components varied with pH (table 2), b u t no significant differences in overall adenosine uptake by human erythrocytes was found at low adenosine concentrations (<2 ~M) between pH 7 and 8. In spite of this, the uptake varied significantly at high adenosine concentrations (>2 pM) and one may speculate as to whether this pH dependence of adenosine uptake is the reason for the differences in vasodilator efficacy of adenosine at different pH observed in vivo: Raberger et al. (1971) administered 1 pg/kg adenosine into one coronary artery of dogs weighing 30 kg with a basal coronary sinus outflow of about 100 ml/min. In these experiments a dose of 30 pg adenosine (dissolved in 1 ml and injected during 1 sec) yielded a plasma concentration of about 50--100 pM disregarding further dilution. This concentration certaintly lies within the concentration range in which the pH dependence of adenosine uptake in erythrocytes was clearly demonstrated in vitro (fig. 2). Altered rates of elimination of adenosine may therefore be a determining factor for the correlation between the vasodilator action of adenosine and the extracellular pH in vivo.
Acknowledgement The expert technical assistance of Miss G. Brugger is appreciated.
pH AND TEMPERATURE DEPENDENCE OF ADENOSINE UPTAKE
References Berlin, R.D., 1973, Temperature dependence of nucleoside membrane transport in rabbit alveolar macrophages and polymorphonuclear leukocytes, J. Biol. Chem. 248, 4724. Boxenbaum, H.G., S. Riegelman and R.M. Elashoff, 1974, Statistical estimations in pharmacokinetics, J. Pharmacokin. Biopharm. 2,123. Neal, J.L., 1972, Analysis of Michaelis kinetics for two independent, saturable membrane transport functions, J. Theor. Biol. 35, 113. Olsson, R.A. and R.E. Patterson, 1976, Adenosine as a physiological regulator of coronary blood flow, Progr. Mol. Subcell. Biol. 4,227. Ottaway, J.H., 1973, Normalization in the fitting of data by iterative methods, Biochem. J. 134,729. Paterson, A.R.P. and J.M. Oliver, 1971, Nucleoside transport. II. Inhibition by p-nitrobenzylthioguanosine and related compounds, Can. J. Biochem. 49,271. Pearson, J.D., J.S. Carleton, A. Hutchings and J.L. Gordon, 1978, Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture, Biochem. J. 170,265. Plagemann, P.G.W. and D.P. Richey, 1974, Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture, Biochim. Biophys. Acta 344,263. Plagemann, P.G.W. and J. Erbe, 1975, Temperaturedependent changes in activation energies of the transport systems for nucleosides, choline and deoxyglucose of cultured Novikoff rat hepatoma cells and effects of cytochalasin B and lipid solvents, J. Membr. Biol. 25,381. Plagemann, P.G.W., R. Marz and J. Erbe 1976, Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff
351
rat hepatoma and mouse L cells: Evidence for a high Km facilitated diffusion system with wide nucleoside specificity, J. Cell. Physiol. 89, 1. Raberger, G., M. Weissel and O. Kraupp, 1971, The dependence of the effects of i. cor. administered adenosine and of coronary conductance on the arterial pH, pCO2 and buffer capacity in dogs, Naunyn-Schmiedeb. Arch. Pharmakol. 271,301. Rauen, H.M., 1964, Biochemisches Taschenbuch, Vol. 2 (Springer Verlag, Berlin/GSttingen/Heidelber) p. 90. Roos, H. and K. Pfleger, 1972, Kinetics of adenosine uptake by erythrocytes, and the influence of dipyridamole, Mol. Pharmacol. 8,417. Schrader, J., R.M. Berne and R. Rubio, 1972, Uptake and metabolism of adenosine by human erythrocyte ghosts, Amer. J. Physiol. 223,159. Sixma, J.J., J.P.M. Lips, A.M.C. Trieschnigg and H. Holmsen, 1976, Transport and metabolism of adenosine in human blood platelets, Biochim. Biophys. Acta 443, 33. Strauss, P.R., J.M. Sheehan and E.R. Kashket, 1976, Membrane transport by routine lymphocytes. I. A rapid sampling technique as applied to the adenosine and thymidine systems, J. Exptl. Med. 144, 1009. Taube, R.A. and R.D. Berlin, 1972, Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes, Biochim. Biophys. Acta 255, 6. Turnheim, K., B. Plank and N. Kolassa, 1978, Inhibition of adenosine uptake in human erythrocytes by adenosine-5'~arboxamides, xylosyladenine, dipyridamole, hexobendine, and p-nitrobenzylthioguanosine, Biochem. Pharmaeol. (in press). Van Belle, H., 1969, Uptake and deamination of adenosine by blood. Species differences, effect of pH, ions, temperature and metabolic inhibitors, Biochim. Biophys. Acta 192,124.