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Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells
Life Sciences, Vol. 43, pp. 1851-1859 Printed in the U.S.A.
Pergamon Press
INTRACELLULAR ADENOSINE FORMATION AND ITS CARRIER-MEDIATED RELEASE IN CULTURED EMBRYONIC CHICK HEART CELLS Parviz Meghji I, Rafael Rubio and Robert M. Berne Department of Physiology, University of Virginia School of Medicine, Charlottesville, VA 22908
(Received in final form October 5, 1988) Summary Adenosine formation and release was examined in 48 hr old primary cultures of chick ventricular myocytes. Dilazep > hexobendine > dipyridamole inhibit incorporation of adenosine into chick embryonic heart cellular nucleotides in a concentration dependent manner. A combination of 30 mM 2deoxyglucose and 2 ~g of oligomycin/ml reduces the ATP content of the cells by 71~ in i0 min. This change is accompanied by an increase in total adenosine concentration of 3.4 nmoles/107cells in i0 min. Although the ATP concentration is not altered during hypoxia (95~N27 / 5~CO2) , adenosine concentration increases by 0.52 nmoles/10 cells in 30 min. When nucleoside incorporation is inhibited by 85-90~ by dipyridamole, dilazep or hexobendine, efflux of adenosine decreases by 70-90~, and 60-90~ of the newly formed adenosine is trapped inside the cells compared to i0~ in the absence of the transport inhibitors, a ,8 -Methylene ADP inhibits the ecto 5'-nucleotidase activity by 91 ± 6~ but does not inhibit adenosine formation or alter its distribution between cells and medium, thus ruling out the involvement of this enzyme in adenosine formation. We conclude that adenosine is formed intracellularly during 2-deoxyglucose and oligomycin-induced ATP degradation and during hypoxia and that the nucleoside is released via the symmetric nucleoside transporter. It has been suggested that adenosine may play a role in restoration of energy balance in the heart and other tissues (1,2). In the heart, adenosine produces vasodilation (I), inhibits the positive inotropic and chronotropic effects exerted by catecholamines both pre- (3) and post-synaptically (4) and decreases heart rate (5). These effects increase substrate and oxygen supply and decrease energy consumption. 5'-Nucleotidase which is mainly responsible for adenosine formation exists in at least two forms, a membrane bound enzyme with its active site on the outside of the plasma membrane (6,7) and a cytosolic 5'-nucleotidase (8) Adenosine release has been explained by three different hypotheses: i. cytoplasmic hydrolysis of AMP followed by outward transport of adenosine via the nucleoside transporter (9); 2. release of AMP or other nucleotides followed by extracellular dephosphorylation by ecto-nucleotidases (10-12); 3. membrane I. Present Address: Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, U.K.
Vascular
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
Biology
Section,
1852
Adenosine Formation and Release
hydrolysis of AMP resulting in outward transport involvement of the nucleoside transporter (13,14).
Vol. 43, No. 2~, 1988
of
adenosine
without
Evidence for the first hypothesis has been obtained using neonatal rat heart cells in culture (15) where nucleoside transport inhibitors reduced adenosine release as well as uptake during 2-deoxyglucose and oligomycininduced ATP catabolism suggesting that adenosine was produced intracellularly and that its efflux occurred via the membrane transporter. Furthermore, the involvement of the ecto-5'-nucleotidase was ruled out using specific inhibitory antibodies (15). On the other hand, Lee et al. (16) have reported that ~,~methylene ADP (AOPCP), an inhibitor of 5'-nucleotidase, eliminated hypoxia induced release of adenosine and accumulated extracellular AMP in freshly isolated embryonic chick heart cells suggesting that nucleotides were transported across the plasma membrane prior to hydrolysis by the ecto-enzyme. This observation together with results showing that transport inhibitors did not effect adenosine release in chick heart cells (17,18) favor hypotheses 2 or 3 and rule out the involvement of the nucleoside transporter in chick heart cells in contrast to rat heart cells. It is necessary to define the cellular site of adenosine production and its mechanism of release for a thorough understanding of coupling between cell metabolism and blood flow. The apparent controversy that arose in studies with hypoxia in chick heart cells (16-18) and metabolic inhibitors (2-deoxyglucose and oligomycin) in neonatal rat heart cells (15) may be a result of the differences in cells used or the differences in methods used to stimulate adenosine formation. Adenosine formation has therefore been examined, in chick heart cells in culture, during 2-deoxyglucose and oligomycin induced reduction in ATP anabolism and during hypoxia. Materials and methods Dipyridamole {2,2',2",2 ....(4,8-dipiperidinopyrimidol[5,4-d]pyrimidine-2,6diyldinitrilo)tetraethanol} was obtained from Boehringer Ingelheim, Bracknell, Berks, U.K. Dilazep {l,4-bis-[3-(3,4,5-trimethoxybenzoyloxy)propyl]perhydro-l, 4 -diazepine} and hexobendine {NN' - dimethyl NN' bis[3-(3',4',5'trimethoxybenzoxy)propyl]ethylene-diamine} were generous gifts from Asta-We~ke, Bielefeld, Germany and Chemic Linzag, Linz, Austria, respectively. 2-[ HIAdenosine and 2-[~H]-AMP were obtained from Amersham, Illinois, USA and a premixed liquid scintillation cocktail for aqueous samples from Beckman, Fullerton, California, USA. Oligomycin, 2-deoxyglucose and ~,~-methylene ADP were obtained from Sigma, St Louis, Missouri, USA. Embryonic chick heart cell cultures Ventricles from about sixty 17 - 18 day old chick embryos were exised, minced and washed thoroughly with ice-cold Mg++/Ca ++ free Hanks' balanced salt solution. Approximately 20 ml of 0.05% trypsin in Hanks' solution were added to the tissue which was gently stirred in a trypsinizing flask for two minutes at room temperature. The supernatant fraction containing isolated cells was then decanted off and fresh trypsin solution was added to the flask and the procedure repeated until most of the tissue was digested. The first two supernatant fractions were discarded and the remainder were collected into precooled 50 ml tubes containing i0 ml of fetal calf serum. The cell suspension was filtered through nylon mesh, washed with and resuspended in a growth medium consisting of 10% fetal calf serum in M~199 medium and plated either in 6 well plates (2 ml/well; ~urface area 960 mm ) or in 24 well plates (0.5 ml/well; surface area 200 mm ). Cells were cultured for 48 hr in an atmosphere of 95% 02/5% CO 2. Prior to use, cells were washed 5 times with Krebs-Ringer bicarbonate/Hepes solution (132 mM NaCI, 13 mM NaHCO3, 13 mM Hepes (N-2-hydroxyethylpiperazine -N' - 2) ethanesulphonic acid/NaOH, pH 7.4),I.3 mM KH2PO 4, 5.3 mM KCI, 1.3 mM MgSO4, 1.4 mM CaCI2) and were then suspended in the same buffer. The cells were estimated to be 70 % myocytes by the selective
Vol. 43, No. 22, 1988
Adenosine Formation and Release
staining method of Blondel et al. (19). largely made up the remaining 30%.
Endothelial
cells
1853
and
fibroblasts
Incorporation of adenosine into nucleotldes Incorporation of [ ~ H ] adenosine (i0 ~M) into cellular nucleotides was measured in the absence and in the presence of adenosine transport inhibitors by the method described previously (15,20). Adenosine formation Rates of adenosine formation may be underestimated in the absence of inhibitors of adenosine metabolism, however preliminary experiments showed that 50 ~M - 5'-amino-5'deoxyadenosine (an adenosine kinase inhibitor) and I0 ~M 2'-deoxycoformycin (an adenosine deaminase inhibitor) did not markedly increase the recovery of adenosine. The inhibitory drugs were therefore not included in further experiments. Adenosine formation, induced by 30 mM 2-deoxyglucose and 2 ~g/ml of oligomycin or by hypoxia, was measured in the absence and presence of nucleoside transport inhibitors or AOPCP. When transport inhibitors or AOPCP were used, cells were preincubated with these drugs for at least 30 min prior to inducing ATP catabolism. Incubations were stopped by removal of 0.9 ml of the supernatant buffer from the wells and addition of this to 0.i ml of 50% (w/v) trichloroacetic acid. Cells were extracted with 1 ml of 5% trichloroacetic acid. Samples were neutralized with 0.5 M tri-n-octylamine in freon mixture and filtered through 0.22 ~m filters. Reverse phase HPLC using a two pump gradient system and a Beckman, Altex Ultrasphere (5 ~m) ODS column (4.6 mm internal diameter X 25 cm) was used for measuring AMP, IMP, adenosine and inosine concentrations. Buffer A was 100 mM KH2PO 4 containing 1% methanol (pH 5.3) and buffer B was i00 mM KH2PO 4 containing 25% methanol (pH 5.57). The gradient profile was linear from 0 to 30% of buffer B in 14 min and was increased to and held at 60% B from 14.1 to 19 min. It was then increased to and held at 100% B from 19.1 min to 23.1 min followed by immediate reversal to 100% of buffer A for column reequilibration. The flow rate was 1.3 ml/min and the total run time was 42 min. ATP and ADP were analyzed using a Partisil i0 SAX (Whatman) anion exchange column with a linear gradient of 5mM (pH 2.8) to 750mM (pH 3.8) NH4H2PO 4 over 36 minutes at a flow rate of 2 ml/min. Peaks were identified and quantified by comparing retention times and peak heights to known samples. When calculating values for metabolites in the cells, a correction was made for the i00 ~i of medium extracted with the cells. Measurement of ecto-5'-nucleotidase activity Ecto-5'-nucleotidase activity was measured as described previously (15) the absence and presence of AOPCP (50 ~M).
in
Cell numbers Cell numbers were estimated by measuring DNA (21) using the value of 2.3 of DNA/cell in chickens (22).
Pg
Cell intactness Cells used in this study were calcium tolerant. The intactness of cells, in the presence of the drugs used and under the experimental conditions used, was judged by measurement of cytosolic lactate dehydrogenase (EC 1.1.1.27) in aspirated supernatants as described by Keiding et al. (23). Values were compared with the activity present in cells lysed with i0 mM Tes/NaOH/O.l% Triton X-IO0. Statistical Methods Means + S.E.M. are given. Differences between groups were tested for significance by Student's paired _t and were not judged to be significant when P > 0.05.
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Adenosine Formation and Release
Vol. 43, No. 23, 1988
Results Adenosine incorporation into cellular nueleotides Adenosine was incorporated into cellular nucleotides at a rate of 74 ± pmol/min per 107 cells (n - 6) in chick heart cells compared to 448 +
12 55
TABLE I Effect Of Adenosine Transport Inhibitors On The Concentrations Of Nucleotides And Nucleosides In Cells And Medium During ATP Catabolism Time ..... MetaboliZes (umol/10" cells)
cells
0 min DOG+ Oligo
i0 min DOG+ Oligo+ Dipyrid
DOG+ Oligo+ Dilazep
DOG+ Oligo+ Hexo
control
control
ATP
28.68± 1.78
30.57± 2.60
8.78±* 2.31
+ 12.39±* 2.63
9.68±* 1.94
9.35±* 2.17
ADP
2.62± 0.18
2.97± 0.22
4.08±*
4.42±*
4.05±*
4.09±*
0.50
0.44
0.38
0.54
ND0.30
ND0.31
2.51±*
2.35±*
2.26±*
2.33±*
0.67
0.66
0.62
0.66
AMP
IMP
adenosine
inosine
medium adenosine
inosine
Total nucleotide and nucleoside concentration
ND0.20
ND0.19
+
+
3.38±*
4.12±*
4.72±*
4.47±*
0.56
0.99
0.89
0.74
+
+
+
1.77±* 0.36 +
2.27±* 0.39 +
2.42±* 0.51 +
7.19±* 1.75 + 0.23±* 0.03 +
8.67±* 1.66 + 0.26±* 0.04 +
8.10±* 1.31 + 0.31±* 0.06 +
ND-
ND-
0.05
0.07
0.39±* 0.12
ND~ 0.08
ND0.19
1.33±* 0.12
ND0.02
ND0.15
2.98±* 0.59
ND0.07
ND0.34
9.01±*
0.10±*
0.11±*
0.15±
1.59
0.02
0.02
0.03
31.50± 1.92
32.78± 2.85
32.45± 1.83
32.37± 2.05
32.03± 2.00
31.22± 1.56
Cells (11.18 X 106 ± 1.90 X 106 ) were incubated at 37°C for 0 min or I0 min in Krebs - Ringer bicarbonate / Hepes solution to which was added 30 m M 2deoxyglucose and 2 ~g of oligomycin / ml and 10 .5 M dipyridamole, 3 X I0 -b M dilazep or 3 X I0- M hexobendine as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for ten different experiments. Medium concentration in nmoles/ml-values given.10-7.number of cells. Abbreviations: DOG, 2-deoxyglucose; Oligo, oligomycin; Dipyrid, dipyridamole; Hexo, hexobendine; ND, not detectable. Nucleotide and nucleoside concentrations in cells and medium of control cells at i0 min were compared with values obtained under different conditions (* P
Vol. 43, No. 23, 1988
Adenosine Formation and Release
1855
pmol/min per 107 in rat heart cells (15). This is consistent with adenosine kinase activity being six fold lower in chicken hearts compared to rat hearts (24). Incorporation of [3H]adenosine (10 .5 M) into cellular nucleotides was inhibited in a concentration dependent manner by ~ipyridamole (EC50=3.1 ± 0.7 X I0-" M, n = 6), d~lazep (EC50=5.0 ± 0.9 X i0 -v M, n = 6) or hexobendine (EC50-1.1 ± 0.4 X i0 -! M, n - 5). Cell intactness was 99.5 ± 0.2%, 99.4 ± 0.1% 98.9 ± 0.4% and 99.2 ± 0.2% in control cells and in the presence of i00 ~M dipyridamole, dilazep, or hexobendine, respectively (n - 6 in each case). Inhibition of 5'-nucleotidase activity Exogenous AMP (180 ~M) was dephosphorylated at a rate of 0.75 ± 0.ii nmol/min per 107 cells (n - 8). The enzyme activity was reduced to 0.07 ± 0.05 TABLE II Effect Of ~ ,8 - Methylene ADP (AOPCP) On The Concentrations Of Nucleotides And Nueleosides In Cells And Medium During ATP Catabolism Time ..... Metabolites (nmol/lO 7 cells)
cells
0 min
I0 min
0 min
I0 min DOG+ Oligo+ AOPCP AOPCP
control
control
DOG+ Oligo
AOPCP
ATP
28.35± 2.54
29.84± 2.55
9.13± 1.28
29.02± 3.03
30.07± 2.51
7.78± 1.70
ADP
2.31± 0.27
2.44± 0.27
4.92± 0.54
2.50± 0.28
2.36± 0.24
4.68± 0.51
AMP
ND0.33
ND0.36
3.30± 0.76
ND0.35
ND0.39
3.17± 0.66
IMP
ND0.15
ND0.i0
2.78± 0.53
ND0.15
ND0.i0
3.11± 0.61
adenosine
ND0.15
ND0.21
0.92± 0.31
ND0.18
ND
1.18± 0.35
inosine
ND
ND
1.48± 0.20
ND
ND0.04
2.21±* 0.31
ND
ND 0.01
3.98± 0.71
ND
ND0.09
4.69± 1.04
ND
ND0.25
4.50± 0.71
ND0.01
ND0.i0
4.94± 0.80
32.51± 2.71
31.01± 1.50
31.67± 3.25
32.59± 2.6
31.76± 2 73
medium adenosine
inosine
Total nucleotide and nucleoside concentration
30.79± 2 79
Cells (8.99 X 106 ± 2.16 X 106 ) were incubated at 37 ° C for 0 min or I0 min in Krebs - Ringer bicarbonate / Hepes solution to which was added 30 mM 2-deoxyglucose and 2 ~g of oligomycin / ml and 5 X 10 .5 M AOPCP as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for seven different experiments. Medium concentration in nmoles/ml~values given.lO-7.number of cells. Abbreviations: DOG, 2-deoxyglucose; AOPCP, ~ ,8 -methylene ADP; Oligo, Oligomycin; ND, not detectable. Values obtained in the presence of AOPCP were compared with those obtained in its absence (*P<0.05).
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Adenosine Formation and Release
nmol/min per 107 (n - 6) in the presence of
Vol. 43, No. 23, 1988
50 pM AOPCP.
Adenosine formation during metabolic poisoning Incubation of cells for i0 min in the absence of drugs did not significantly alter the ATP concentration. However, when the cells were incubated with a combination of 30 mM 2-deoxyglucose and 2 ~g of oligomycin/ml there was a 71 % decrease in ATP concentrations in I0 min (Table I). Nucleotides could not be detected in the medium. The decrease in ATP concentration was accounted for by ADP (5.1%), AMP (11.5%), IMP (15.5%), adenosine (15.5%) and inosine (47.4%). After i0 min of incubation, 12 % of the adenosine formed was found inside the TABLE III Effect Of Adenosine Transport Inhibitors On The Concentrations Of Nucleotides And Nucleosides In Cells And Medium During hypoxia Time ..... MetaboliZes (nmol/10" cells) cells
0 min
30 min
(95% N2 and 5% C02)
control
control
AOPCP
Dipyrid
Dilazep
Hexo
ATP
28.48± 1.68
31.13± 2.99
30.39± 4.17
32.71± 2.57
32.89± 2.87
31.91± 2.99
ADP
2.50± 0.27
2.83± 0.62
2.45± 0.30
2.53± 0.32
2.61± 0.99
2.45± 0.34
AMP
ND0.15
ND0.24
ND0.13
ND0.28
ND0.25
ND0.30
IMP
0.24± 0.06
0.42± 0.09
0.36± 0.09
0.46± 0.12
0.43± 0.07
0.39± 0.08
adenosine
ND 0.06
0.05± 0.02
0.04± 0.02
0.20±* 0.09
0.26± 0.13
0.20±* 0.08
inosine
ND 0.01
0.13± 0.03
0.13± 0.04
0.74±* 0.23
0.91±* 0.21
0.93±* 0.21
ND 0.003
0.47± 0.15
0.56± 0.14
0.11±*
0.13±*
0.13±*
0.03
0.05
0.04
ND
0.68± 0.09
0.88± 0.14
0.08±*
0.13±*
0.19±*
0.02
0.03
0.05
35.64± 2.80
34.87± 3.25
36.91± 2.91
37.45± 3.26
36.29± 3.14
medium adenosine
inosine
Total nucleotide and nucleoside concentration
-
31.31± 1.79
Cells (6.73 X 106 ± 0.79 X 106) were incubated at 37°C for 0 min or 30 min in Krebs - Ringer bicarbonate / Hepes solution in an atmosphere of 95% N 2 and 5% CO 2 in th~ absence and presence of 5 X 10 .5 M AOPCP, 10 .5 M dipyridamole, 3 X 10-VM dilazep or 3 X i0 "U M hexobendine as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for si~ different experiments. Medium concentration in nmoles/ml-values given.10- .number of cells. Abbreviations: Dipyrid, dipyridamole; Hexo, hexobendine; ND, not detectable. Nucleotide and nucleoside concentrations in cells and medium of cells incubated for 30 min in the absence of drugs were compared with values obtained in the presence of AOPCP, dipyridamole, dilazep or hexobendine (* P<0.05).
Vol. 43, No. 23, 1988
Adenosine Formation and Release
1857
cells and the remainder in the medium. Dipyridamole (i0 -b M), dilazep (3 X 10"6M) and hexobendine (3 X 10 -6 M) at concentrations that inhibited incorporation of adenosine into cellular nucleotides by 84.7 + 2.5 %, 90.0 ± 1.0 % and 88.8 ± 0.7 %, increased the amount of adenosine (nmoles/107 cells) inside the cells from 0.39 ± 0.12 to 1.77 ± 0.36, 2.27 ± 0.39 and 2.42 ± 0.51 (Table I), respectively. 7 This was accompanied by a reduction in adenosine concentration (nmoles/10 cells) in the medium from 2.98 ± 0.59 to 0.23 ± 0.03 in the presence of dipyridamole, 0.26 ± 0.04 in the presence of dilazep and 0.31 ± 0.06 in the presence of hexobendlne (Table I). The effects of transport inhibitor on inosine distribution followed a similar pattern as that described for adenosine (Table I). The total nucleoside and nucleotide concentrations under the various conditions did not significantly differ from that of control cells at I0 min (Table I). Cells remained at 99.2 ± 0.4% of intact levels at 0 min, 99.5 ± 0.1% at i0 min and 99.2 ± 0.2% at i0 min in the presence of 2-deoxyglucose and oligomycin. In the additional presence of dipyridamole, dilazep and hexobendlne, release of lactate dehydrogenase indicated that the cells were 99.4 ± 0 . 1 % , 99.3 ± 0 . 1 % and 99.2 ± 0.2 % of the values observed in intact controls, respectively (n - I0 in each case). Incubation with AOPCP did not result in an increase in AMP accumulation in cells or medium nor did it alter the concentration of adenosine in cells or media (Table II). Nor were any of the other metabolites altered with exception of inosine in the cells, which increased significantly in the presence of AOPCP (Table II). Cells were 99.6 ± 0 . 1 % intact in the presence of AOPCP (n -6). Adenosine formation during hvpoxia Incubation of cells for 30 min in an atmosphere of 95% N2/5%C02 did not alter the ATP concentration in the cells. ATP or other nucleotides could not be detected ~n the medium. However the adenosine ~oncentration increased by 0.52 nmoles/10" cells and inosine by 0.81 nmoles/10 cells in 32 min in cells and medium (T~ble III). Dipyridamole (IO-5M), dilazep (3 X i0- M) and hexobendine (3 X 10-VM), at concentrations that inhibited incorporation of adenosine into cellular nucleotides by 85-90%, increased adenosine concentration (nmoles/107 cells) inside the cells from 0.05 ± 0.02 to 0.20 ± 0.09, 0.26 ± 0.13 and 0.20 ± 0.08, respectively (Table III). T h i s was accompanied by a reduction in adenosine concentration (nmoles/10 / cells) in the medium from 0.47 ± 0.15 to 0.Ii ± 0.03 in the presence of dipyridamole, 0.13 ± 0.05 in the presence of dilazep and 0.13 ± 0.04 in the presence of hexobendine (Table III). The effects of transport inhibitors, on inosine distribution followed a similar pattern as that described for adenosine (Table III). Incubation with AOPCP did not alter the concentration of adenosine in cells or media (Table III). Cells were 99.6 ± 0.1% intact at 0 min and 99.6% ± 0.1% at 30 min. In the additional presence of AOPCP, dipyridamole, dilazep or hexobendine cells were 99.5 ± 0.2%, 99.4 ± 0.1%, 99.4 ± 0.3% and 99.4 ± 0.1% intact, respectively. Discussion Nucleoside transport inhibitors, dipyridamole, hexobendine and dilazep inhibited incorporation of adenosine into cellular nucleotides in cultured chick embryonic heart cells with EC50 values which were comparable to those obtained previously (18) except in the case of dipyridamole which was ten times less potent in our study. The reasons for this difference is not known. Dipyridamole, hexobendine and dilazep at concentrations that inhibited incorporation of adenosine by 85 - 90%, increased accumulation of adenosine inside the cells from 10% to 60 - 90% and decreased efflux of adenosine by 70 90%, suggesting that adenosine is produced intracellularly and that its efflux occurs via the nucleoside transporter. Dipyridamole, dilazep and hexobendine
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Adenosine Formation and Release
Vol. 43, No. 23, 1988
also increased the amount of inosine in the cells from 13% to 98-99% and decreased that in the medium by 98-99%. This is consistent with inosine being exported out of the cell via the same transporter as adenosine (25). Nucleoside transport inhibitors have been shown to affect both release and uptake of adenosine in perfused hearts (9,26,27), in cultured neonatal rat heart cells (15) and freshly isolated hepatocytes (28). This is consistent with directionally symmetrical transport of adenosine (25,29). In contrast, Mustafa et al. (17), Mustafa (18) and Lee et al. (30) have reported the failure of dipyridamole, dilazep, hexobendine, lidoflazine and papaverine to inhibit adenosine release from embryonic chick heart cells. This conflict in results may be explained by the differences in methodology used to separate cells from medium. Some contamination of medium with cells would be inevitable with the method used by Mustafa et al, (17) and Mustafa (18) where incubations were terminated by scraping the cells from the culture dishes with a plastic spatula and separating them from the medium by centrifugation. In our hands this procedure resulted in 32.8 ± 4.7% cell breakage (n-7) compared to 0.39 ± 0.06% (n=7) when the medium was aspirated off and the cells separately extracted as in the present study. The method used by Mustafa et al. (17) and Mustafa (18) also resulted in leakage of adenine nucleotides into the medium thus exposing them to ecto-5'-nucleotidase and presumably there was also leakage of the accumulated intracellular nucleosides into the medium. An asymmetric effect of nucleoside inhibitors on release and uptake, however, has also been demonstrated in cultured aortic endothelial cells from pig (31) and cultured aortic smooth muscle cells from rat (32). Thus, although the possibilty of differences between cell types may exist, this does not appear to be the case between embryonic chick and neonate rat heart cells. In this study as in a previous study (15), nucleotides could not be detected in the medium. Furthermore, the concentrations of AMP in cells or medium were not altered by inhibition of the ecto-enzyme by AOPCP as would be expected if adenosine were being formed extracellularly or if the ecto- enzyme were acting as a translocase. In addition, inhibition of 5'-nucleotidase failed to prevent the production or release of adenosine from cultured chick embryonic heart cells in the present study, a finding consistent with other studies on neonatal rat heart cells (15), perfused hearts (9,33), isolated hepatocytes (28), brain slices (34), and rat polymorphonuclear leukocytes (35). Lee et al. (16), however, have reported that AOPCP eliminated the hypoxia induced release of adenosine in freshly isolated embryonic chick heart cells which resulted in extracellular accumulation of AMP. This discrepancy may be due to cell breakage that might be expected in freshly isolated cells. However, release of AMP and accumulation of AMP in the presence of AOPCP have also been demonstrated in perfused hearts (36). The present study demonstrates that adenosine formation in embryonic chick heart cells occurs via the same mechanism as described previously for neonatal rat heart cells (15) in which adenosine is formed intracellularly and is exported from the cell via the nucleoside transporter. Acknowledgements We gratefully acknowledge the travel grant received by Parviz Meghji from the Wellcome Trust in the U.K. and the expert technical assistance of Luvenia Wigginton and Mia Taylor. Supported by NIH grants HL-I0384 and HI-19242. References I. R. M. BERNE, Am. J. Physiol. 204, 317-322 (1963). 2. A.C. NEWBY, Trends Biochem. Sci. 9, 42-44 (1984). 3. P. HEDQVlST and B.B. FREDHOLM, Acta Physiol. Scand. 105, 120-122 (1979).
Vol. 43, No. 23, 1988
4. 5. 6 7. 8. 9. I0. Ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
Adenosine Formation and Release
1859
J. SCHRADER, G. BAUMANN and E. GERLACH, Pflugers Arch. 372, 29-35 (1977). T.N. JAMES, J. Pharmacol. Exp. Ther. 149, 233-247 (1965). R. RUBIO, R.M. BERNE and J.G. DOBSON, Am. J. Physiol. 225, 938-953 (1973). M.D. BARON, B. POPE and J.P. LUZIO, Biochem. J. 236, 495-502 (1986). R. ITOH, J. OKA and H. OZASA, Biochem. J. 235, 847-851 (1986). W. SCHUTZ, J. SCHRADER and E. GERLACH, Am. J. Physiol. 240, H963-970. (1981). B.M. PADDLE and O. BURNSTOCK, Blood Vessels ii, 110-119 (1974). T. FORRESTER and C.A. WILLIAMS, J. Physiol. (London) 268, 371-390 (1977). J.D. PEARSON, J.S. CARLETON and J.L. GORDON, Biochem. J. 190, 421-429 (1980). J.R.S. ARCH and E.A. NEWSHOLME, Essays Biochem. 14, 82-123 (1978). G.P. FRICK and LOWENSTEIN J.M., J. Biol. Chem. 253, 1240-1244 (1978). P. MEGHJI, C.A. HOLMQUIST & A.C. NEWBY, Biochem. J. 229, 799-805 (1985). B.K. LEE, R. RUBIO and R.M. BERNE, Circulation, 70, (SuppII)-83 (1984a). S.J. MUSTAFA, R.M. BERNE and R. RUBIO, Am. J. Physiol. 228, 1474-1478 (1975). S.J. MUSTAFA, Biochem. Pharmacol. 28 , 2617-2624 (1979). B. BLONDEL, I. ROIJEN and J.P. CHENEVAL, Experientia 27, 356-358 (1971). A.C. NEWBY, C.A. HOLMQUIST, J. ILLINGWORTH and J.D. PEARSON, Biochem. J. 214, 317-323 (1983). J.M. KISSANE and E. ROBINS, J. Biol. Chem. 233, 184-188 (1958). H. REES and R.N. JONES, Int. Rev. Cytology 32, 53-92 (1972). R. KEIDING, M. HORDER, W. GERHARDT, E. PITKANEN, R. TENHUNEN, J.H. STROMME, L. THEODORSEN, J. WALDENSTROM, N. TRYDING and L. WESTLUND, Scan. J. Clin. Lab. Invest. 33, 291-306 (1974). J.R.S. ARCH and E.A. NEWSHOLME, Biochem. J. 174, 965-977 (1978) P.G.W. PLAGEMANN and R.M. WOHLHEUTER, In Regulatory Function of Adenosine (Berne, R. M., Rall, T. W. & Rubio, R., eds.), pp. 179-201, Martinus Nijhoff Publishers, The Hague, Boston and London (1983). F. H. DECENRINC, R.R. CURNISH, R. RUBIO and R.M. BERNE, J. Mol. Cell. Cardiol. 8, 877-888 (1976). J.C. DOBSON and J. SCHRADER, J. Mol. Cell. Cardiol. 16, 813-822 (1984). F.L. BELLONI, P.L. ELKIN and B. CIANNOTTO, Br. J. Pharmacol., 85, 441-446 (1985). A.R.P. PATERSON, N. KOLASSA, and C.E. CASS Pharmacol. Ther., 12, 515-536 (1981). B.K. LEE, R. RUBIO and R.M. BERNE, Fed. Proc. 43, 307 (1984b). J.C. SHRYOCK and R.M. BERNE, Microvasc. Res. 27, 263 (1984). F.L. BELLONI, S.P. BRUTTIG, R. RUBIO and R. M. BERNE, Microvascular Res. 32, 200-210 (1986). G.P. FRICK and J.M. LOWENSTEIN, J. Biol. Chem. 251, 6372-6378 (1976). F. PONS, R.F. BRUNS and J.W. DALY, J. Neurochem. 34, 1319-1323 (1980). A.C. NEWBY, and C.A. HOLMQUIST, Biochem. J. 200, 399-403 (1981). S. IMAI, M. NAKAZAWA, H. IMAI and H. JIN, In Topics and Perspectives in Adenosine Research, pp. 416-424, Springer-Verlag, Berlin, Heidelberg, New York, London, Paris, Tokyo (1987).
Pergamon Press
INTRACELLULAR ADENOSINE FORMATION AND ITS CARRIER-MEDIATED RELEASE IN CULTURED EMBRYONIC CHICK HEART CELLS Parviz Meghji I, Rafael Rubio and Robert M. Berne Department of Physiology, University of Virginia School of Medicine, Charlottesville, VA 22908
(Received in final form October 5, 1988) Summary Adenosine formation and release was examined in 48 hr old primary cultures of chick ventricular myocytes. Dilazep > hexobendine > dipyridamole inhibit incorporation of adenosine into chick embryonic heart cellular nucleotides in a concentration dependent manner. A combination of 30 mM 2deoxyglucose and 2 ~g of oligomycin/ml reduces the ATP content of the cells by 71~ in i0 min. This change is accompanied by an increase in total adenosine concentration of 3.4 nmoles/107cells in i0 min. Although the ATP concentration is not altered during hypoxia (95~N27 / 5~CO2) , adenosine concentration increases by 0.52 nmoles/10 cells in 30 min. When nucleoside incorporation is inhibited by 85-90~ by dipyridamole, dilazep or hexobendine, efflux of adenosine decreases by 70-90~, and 60-90~ of the newly formed adenosine is trapped inside the cells compared to i0~ in the absence of the transport inhibitors, a ,8 -Methylene ADP inhibits the ecto 5'-nucleotidase activity by 91 ± 6~ but does not inhibit adenosine formation or alter its distribution between cells and medium, thus ruling out the involvement of this enzyme in adenosine formation. We conclude that adenosine is formed intracellularly during 2-deoxyglucose and oligomycin-induced ATP degradation and during hypoxia and that the nucleoside is released via the symmetric nucleoside transporter. It has been suggested that adenosine may play a role in restoration of energy balance in the heart and other tissues (1,2). In the heart, adenosine produces vasodilation (I), inhibits the positive inotropic and chronotropic effects exerted by catecholamines both pre- (3) and post-synaptically (4) and decreases heart rate (5). These effects increase substrate and oxygen supply and decrease energy consumption. 5'-Nucleotidase which is mainly responsible for adenosine formation exists in at least two forms, a membrane bound enzyme with its active site on the outside of the plasma membrane (6,7) and a cytosolic 5'-nucleotidase (8) Adenosine release has been explained by three different hypotheses: i. cytoplasmic hydrolysis of AMP followed by outward transport of adenosine via the nucleoside transporter (9); 2. release of AMP or other nucleotides followed by extracellular dephosphorylation by ecto-nucleotidases (10-12); 3. membrane I. Present Address: Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, U.K.
Vascular
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
Biology
Section,
1852
Adenosine Formation and Release
hydrolysis of AMP resulting in outward transport involvement of the nucleoside transporter (13,14).
Vol. 43, No. 2~, 1988
of
adenosine
without
Evidence for the first hypothesis has been obtained using neonatal rat heart cells in culture (15) where nucleoside transport inhibitors reduced adenosine release as well as uptake during 2-deoxyglucose and oligomycininduced ATP catabolism suggesting that adenosine was produced intracellularly and that its efflux occurred via the membrane transporter. Furthermore, the involvement of the ecto-5'-nucleotidase was ruled out using specific inhibitory antibodies (15). On the other hand, Lee et al. (16) have reported that ~,~methylene ADP (AOPCP), an inhibitor of 5'-nucleotidase, eliminated hypoxia induced release of adenosine and accumulated extracellular AMP in freshly isolated embryonic chick heart cells suggesting that nucleotides were transported across the plasma membrane prior to hydrolysis by the ecto-enzyme. This observation together with results showing that transport inhibitors did not effect adenosine release in chick heart cells (17,18) favor hypotheses 2 or 3 and rule out the involvement of the nucleoside transporter in chick heart cells in contrast to rat heart cells. It is necessary to define the cellular site of adenosine production and its mechanism of release for a thorough understanding of coupling between cell metabolism and blood flow. The apparent controversy that arose in studies with hypoxia in chick heart cells (16-18) and metabolic inhibitors (2-deoxyglucose and oligomycin) in neonatal rat heart cells (15) may be a result of the differences in cells used or the differences in methods used to stimulate adenosine formation. Adenosine formation has therefore been examined, in chick heart cells in culture, during 2-deoxyglucose and oligomycin induced reduction in ATP anabolism and during hypoxia. Materials and methods Dipyridamole {2,2',2",2 ....(4,8-dipiperidinopyrimidol[5,4-d]pyrimidine-2,6diyldinitrilo)tetraethanol} was obtained from Boehringer Ingelheim, Bracknell, Berks, U.K. Dilazep {l,4-bis-[3-(3,4,5-trimethoxybenzoyloxy)propyl]perhydro-l, 4 -diazepine} and hexobendine {NN' - dimethyl NN' bis[3-(3',4',5'trimethoxybenzoxy)propyl]ethylene-diamine} were generous gifts from Asta-We~ke, Bielefeld, Germany and Chemic Linzag, Linz, Austria, respectively. 2-[ HIAdenosine and 2-[~H]-AMP were obtained from Amersham, Illinois, USA and a premixed liquid scintillation cocktail for aqueous samples from Beckman, Fullerton, California, USA. Oligomycin, 2-deoxyglucose and ~,~-methylene ADP were obtained from Sigma, St Louis, Missouri, USA. Embryonic chick heart cell cultures Ventricles from about sixty 17 - 18 day old chick embryos were exised, minced and washed thoroughly with ice-cold Mg++/Ca ++ free Hanks' balanced salt solution. Approximately 20 ml of 0.05% trypsin in Hanks' solution were added to the tissue which was gently stirred in a trypsinizing flask for two minutes at room temperature. The supernatant fraction containing isolated cells was then decanted off and fresh trypsin solution was added to the flask and the procedure repeated until most of the tissue was digested. The first two supernatant fractions were discarded and the remainder were collected into precooled 50 ml tubes containing i0 ml of fetal calf serum. The cell suspension was filtered through nylon mesh, washed with and resuspended in a growth medium consisting of 10% fetal calf serum in M~199 medium and plated either in 6 well plates (2 ml/well; ~urface area 960 mm ) or in 24 well plates (0.5 ml/well; surface area 200 mm ). Cells were cultured for 48 hr in an atmosphere of 95% 02/5% CO 2. Prior to use, cells were washed 5 times with Krebs-Ringer bicarbonate/Hepes solution (132 mM NaCI, 13 mM NaHCO3, 13 mM Hepes (N-2-hydroxyethylpiperazine -N' - 2) ethanesulphonic acid/NaOH, pH 7.4),I.3 mM KH2PO 4, 5.3 mM KCI, 1.3 mM MgSO4, 1.4 mM CaCI2) and were then suspended in the same buffer. The cells were estimated to be 70 % myocytes by the selective
Vol. 43, No. 22, 1988
Adenosine Formation and Release
staining method of Blondel et al. (19). largely made up the remaining 30%.
Endothelial
cells
1853
and
fibroblasts
Incorporation of adenosine into nucleotldes Incorporation of [ ~ H ] adenosine (i0 ~M) into cellular nucleotides was measured in the absence and in the presence of adenosine transport inhibitors by the method described previously (15,20). Adenosine formation Rates of adenosine formation may be underestimated in the absence of inhibitors of adenosine metabolism, however preliminary experiments showed that 50 ~M - 5'-amino-5'deoxyadenosine (an adenosine kinase inhibitor) and I0 ~M 2'-deoxycoformycin (an adenosine deaminase inhibitor) did not markedly increase the recovery of adenosine. The inhibitory drugs were therefore not included in further experiments. Adenosine formation, induced by 30 mM 2-deoxyglucose and 2 ~g/ml of oligomycin or by hypoxia, was measured in the absence and presence of nucleoside transport inhibitors or AOPCP. When transport inhibitors or AOPCP were used, cells were preincubated with these drugs for at least 30 min prior to inducing ATP catabolism. Incubations were stopped by removal of 0.9 ml of the supernatant buffer from the wells and addition of this to 0.i ml of 50% (w/v) trichloroacetic acid. Cells were extracted with 1 ml of 5% trichloroacetic acid. Samples were neutralized with 0.5 M tri-n-octylamine in freon mixture and filtered through 0.22 ~m filters. Reverse phase HPLC using a two pump gradient system and a Beckman, Altex Ultrasphere (5 ~m) ODS column (4.6 mm internal diameter X 25 cm) was used for measuring AMP, IMP, adenosine and inosine concentrations. Buffer A was 100 mM KH2PO 4 containing 1% methanol (pH 5.3) and buffer B was i00 mM KH2PO 4 containing 25% methanol (pH 5.57). The gradient profile was linear from 0 to 30% of buffer B in 14 min and was increased to and held at 60% B from 14.1 to 19 min. It was then increased to and held at 100% B from 19.1 min to 23.1 min followed by immediate reversal to 100% of buffer A for column reequilibration. The flow rate was 1.3 ml/min and the total run time was 42 min. ATP and ADP were analyzed using a Partisil i0 SAX (Whatman) anion exchange column with a linear gradient of 5mM (pH 2.8) to 750mM (pH 3.8) NH4H2PO 4 over 36 minutes at a flow rate of 2 ml/min. Peaks were identified and quantified by comparing retention times and peak heights to known samples. When calculating values for metabolites in the cells, a correction was made for the i00 ~i of medium extracted with the cells. Measurement of ecto-5'-nucleotidase activity Ecto-5'-nucleotidase activity was measured as described previously (15) the absence and presence of AOPCP (50 ~M).
in
Cell numbers Cell numbers were estimated by measuring DNA (21) using the value of 2.3 of DNA/cell in chickens (22).
Pg
Cell intactness Cells used in this study were calcium tolerant. The intactness of cells, in the presence of the drugs used and under the experimental conditions used, was judged by measurement of cytosolic lactate dehydrogenase (EC 1.1.1.27) in aspirated supernatants as described by Keiding et al. (23). Values were compared with the activity present in cells lysed with i0 mM Tes/NaOH/O.l% Triton X-IO0. Statistical Methods Means + S.E.M. are given. Differences between groups were tested for significance by Student's paired _t and were not judged to be significant when P > 0.05.
1854
Adenosine Formation and Release
Vol. 43, No. 23, 1988
Results Adenosine incorporation into cellular nueleotides Adenosine was incorporated into cellular nucleotides at a rate of 74 ± pmol/min per 107 cells (n - 6) in chick heart cells compared to 448 +
12 55
TABLE I Effect Of Adenosine Transport Inhibitors On The Concentrations Of Nucleotides And Nucleosides In Cells And Medium During ATP Catabolism Time ..... MetaboliZes (umol/10" cells)
cells
0 min DOG+ Oligo
i0 min DOG+ Oligo+ Dipyrid
DOG+ Oligo+ Dilazep
DOG+ Oligo+ Hexo
control
control
ATP
28.68± 1.78
30.57± 2.60
8.78±* 2.31
+ 12.39±* 2.63
9.68±* 1.94
9.35±* 2.17
ADP
2.62± 0.18
2.97± 0.22
4.08±*
4.42±*
4.05±*
4.09±*
0.50
0.44
0.38
0.54
ND0.30
ND0.31
2.51±*
2.35±*
2.26±*
2.33±*
0.67
0.66
0.62
0.66
AMP
IMP
adenosine
inosine
medium adenosine
inosine
Total nucleotide and nucleoside concentration
ND0.20
ND0.19
+
+
3.38±*
4.12±*
4.72±*
4.47±*
0.56
0.99
0.89
0.74
+
+
+
1.77±* 0.36 +
2.27±* 0.39 +
2.42±* 0.51 +
7.19±* 1.75 + 0.23±* 0.03 +
8.67±* 1.66 + 0.26±* 0.04 +
8.10±* 1.31 + 0.31±* 0.06 +
ND-
ND-
0.05
0.07
0.39±* 0.12
ND~ 0.08
ND0.19
1.33±* 0.12
ND0.02
ND0.15
2.98±* 0.59
ND0.07
ND0.34
9.01±*
0.10±*
0.11±*
0.15±
1.59
0.02
0.02
0.03
31.50± 1.92
32.78± 2.85
32.45± 1.83
32.37± 2.05
32.03± 2.00
31.22± 1.56
Cells (11.18 X 106 ± 1.90 X 106 ) were incubated at 37°C for 0 min or I0 min in Krebs - Ringer bicarbonate / Hepes solution to which was added 30 m M 2deoxyglucose and 2 ~g of oligomycin / ml and 10 .5 M dipyridamole, 3 X I0 -b M dilazep or 3 X I0- M hexobendine as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for ten different experiments. Medium concentration in nmoles/ml-values given.10-7.number of cells. Abbreviations: DOG, 2-deoxyglucose; Oligo, oligomycin; Dipyrid, dipyridamole; Hexo, hexobendine; ND, not detectable. Nucleotide and nucleoside concentrations in cells and medium of control cells at i0 min were compared with values obtained under different conditions (* P
Vol. 43, No. 23, 1988
Adenosine Formation and Release
1855
pmol/min per 107 in rat heart cells (15). This is consistent with adenosine kinase activity being six fold lower in chicken hearts compared to rat hearts (24). Incorporation of [3H]adenosine (10 .5 M) into cellular nucleotides was inhibited in a concentration dependent manner by ~ipyridamole (EC50=3.1 ± 0.7 X I0-" M, n = 6), d~lazep (EC50=5.0 ± 0.9 X i0 -v M, n = 6) or hexobendine (EC50-1.1 ± 0.4 X i0 -! M, n - 5). Cell intactness was 99.5 ± 0.2%, 99.4 ± 0.1% 98.9 ± 0.4% and 99.2 ± 0.2% in control cells and in the presence of i00 ~M dipyridamole, dilazep, or hexobendine, respectively (n - 6 in each case). Inhibition of 5'-nucleotidase activity Exogenous AMP (180 ~M) was dephosphorylated at a rate of 0.75 ± 0.ii nmol/min per 107 cells (n - 8). The enzyme activity was reduced to 0.07 ± 0.05 TABLE II Effect Of ~ ,8 - Methylene ADP (AOPCP) On The Concentrations Of Nucleotides And Nueleosides In Cells And Medium During ATP Catabolism Time ..... Metabolites (nmol/lO 7 cells)
cells
0 min
I0 min
0 min
I0 min DOG+ Oligo+ AOPCP AOPCP
control
control
DOG+ Oligo
AOPCP
ATP
28.35± 2.54
29.84± 2.55
9.13± 1.28
29.02± 3.03
30.07± 2.51
7.78± 1.70
ADP
2.31± 0.27
2.44± 0.27
4.92± 0.54
2.50± 0.28
2.36± 0.24
4.68± 0.51
AMP
ND0.33
ND0.36
3.30± 0.76
ND0.35
ND0.39
3.17± 0.66
IMP
ND0.15
ND0.i0
2.78± 0.53
ND0.15
ND0.i0
3.11± 0.61
adenosine
ND0.15
ND0.21
0.92± 0.31
ND0.18
ND
1.18± 0.35
inosine
ND
ND
1.48± 0.20
ND
ND0.04
2.21±* 0.31
ND
ND 0.01
3.98± 0.71
ND
ND0.09
4.69± 1.04
ND
ND0.25
4.50± 0.71
ND0.01
ND0.i0
4.94± 0.80
32.51± 2.71
31.01± 1.50
31.67± 3.25
32.59± 2.6
31.76± 2 73
medium adenosine
inosine
Total nucleotide and nucleoside concentration
30.79± 2 79
Cells (8.99 X 106 ± 2.16 X 106 ) were incubated at 37 ° C for 0 min or I0 min in Krebs - Ringer bicarbonate / Hepes solution to which was added 30 mM 2-deoxyglucose and 2 ~g of oligomycin / ml and 5 X 10 .5 M AOPCP as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for seven different experiments. Medium concentration in nmoles/ml~values given.lO-7.number of cells. Abbreviations: DOG, 2-deoxyglucose; AOPCP, ~ ,8 -methylene ADP; Oligo, Oligomycin; ND, not detectable. Values obtained in the presence of AOPCP were compared with those obtained in its absence (*P<0.05).
1856
Adenosine Formation and Release
nmol/min per 107 (n - 6) in the presence of
Vol. 43, No. 23, 1988
50 pM AOPCP.
Adenosine formation during metabolic poisoning Incubation of cells for i0 min in the absence of drugs did not significantly alter the ATP concentration. However, when the cells were incubated with a combination of 30 mM 2-deoxyglucose and 2 ~g of oligomycin/ml there was a 71 % decrease in ATP concentrations in I0 min (Table I). Nucleotides could not be detected in the medium. The decrease in ATP concentration was accounted for by ADP (5.1%), AMP (11.5%), IMP (15.5%), adenosine (15.5%) and inosine (47.4%). After i0 min of incubation, 12 % of the adenosine formed was found inside the TABLE III Effect Of Adenosine Transport Inhibitors On The Concentrations Of Nucleotides And Nucleosides In Cells And Medium During hypoxia Time ..... MetaboliZes (nmol/10" cells) cells
0 min
30 min
(95% N2 and 5% C02)
control
control
AOPCP
Dipyrid
Dilazep
Hexo
ATP
28.48± 1.68
31.13± 2.99
30.39± 4.17
32.71± 2.57
32.89± 2.87
31.91± 2.99
ADP
2.50± 0.27
2.83± 0.62
2.45± 0.30
2.53± 0.32
2.61± 0.99
2.45± 0.34
AMP
ND0.15
ND0.24
ND0.13
ND0.28
ND0.25
ND0.30
IMP
0.24± 0.06
0.42± 0.09
0.36± 0.09
0.46± 0.12
0.43± 0.07
0.39± 0.08
adenosine
ND 0.06
0.05± 0.02
0.04± 0.02
0.20±* 0.09
0.26± 0.13
0.20±* 0.08
inosine
ND 0.01
0.13± 0.03
0.13± 0.04
0.74±* 0.23
0.91±* 0.21
0.93±* 0.21
ND 0.003
0.47± 0.15
0.56± 0.14
0.11±*
0.13±*
0.13±*
0.03
0.05
0.04
ND
0.68± 0.09
0.88± 0.14
0.08±*
0.13±*
0.19±*
0.02
0.03
0.05
35.64± 2.80
34.87± 3.25
36.91± 2.91
37.45± 3.26
36.29± 3.14
medium adenosine
inosine
Total nucleotide and nucleoside concentration
-
31.31± 1.79
Cells (6.73 X 106 ± 0.79 X 106) were incubated at 37°C for 0 min or 30 min in Krebs - Ringer bicarbonate / Hepes solution in an atmosphere of 95% N 2 and 5% CO 2 in th~ absence and presence of 5 X 10 .5 M AOPCP, 10 .5 M dipyridamole, 3 X 10-VM dilazep or 3 X i0 "U M hexobendine as indicated. Metabolites were measured in duplicate. The values given are means ± S.E.M. for si~ different experiments. Medium concentration in nmoles/ml-values given.10- .number of cells. Abbreviations: Dipyrid, dipyridamole; Hexo, hexobendine; ND, not detectable. Nucleotide and nucleoside concentrations in cells and medium of cells incubated for 30 min in the absence of drugs were compared with values obtained in the presence of AOPCP, dipyridamole, dilazep or hexobendine (* P<0.05).
Vol. 43, No. 23, 1988
Adenosine Formation and Release
1857
cells and the remainder in the medium. Dipyridamole (i0 -b M), dilazep (3 X 10"6M) and hexobendine (3 X 10 -6 M) at concentrations that inhibited incorporation of adenosine into cellular nucleotides by 84.7 + 2.5 %, 90.0 ± 1.0 % and 88.8 ± 0.7 %, increased the amount of adenosine (nmoles/107 cells) inside the cells from 0.39 ± 0.12 to 1.77 ± 0.36, 2.27 ± 0.39 and 2.42 ± 0.51 (Table I), respectively. 7 This was accompanied by a reduction in adenosine concentration (nmoles/10 cells) in the medium from 2.98 ± 0.59 to 0.23 ± 0.03 in the presence of dipyridamole, 0.26 ± 0.04 in the presence of dilazep and 0.31 ± 0.06 in the presence of hexobendlne (Table I). The effects of transport inhibitor on inosine distribution followed a similar pattern as that described for adenosine (Table I). The total nucleoside and nucleotide concentrations under the various conditions did not significantly differ from that of control cells at I0 min (Table I). Cells remained at 99.2 ± 0.4% of intact levels at 0 min, 99.5 ± 0.1% at i0 min and 99.2 ± 0.2% at i0 min in the presence of 2-deoxyglucose and oligomycin. In the additional presence of dipyridamole, dilazep and hexobendlne, release of lactate dehydrogenase indicated that the cells were 99.4 ± 0 . 1 % , 99.3 ± 0 . 1 % and 99.2 ± 0.2 % of the values observed in intact controls, respectively (n - I0 in each case). Incubation with AOPCP did not result in an increase in AMP accumulation in cells or medium nor did it alter the concentration of adenosine in cells or media (Table II). Nor were any of the other metabolites altered with exception of inosine in the cells, which increased significantly in the presence of AOPCP (Table II). Cells were 99.6 ± 0 . 1 % intact in the presence of AOPCP (n -6). Adenosine formation during hvpoxia Incubation of cells for 30 min in an atmosphere of 95% N2/5%C02 did not alter the ATP concentration in the cells. ATP or other nucleotides could not be detected ~n the medium. However the adenosine ~oncentration increased by 0.52 nmoles/10" cells and inosine by 0.81 nmoles/10 cells in 32 min in cells and medium (T~ble III). Dipyridamole (IO-5M), dilazep (3 X i0- M) and hexobendine (3 X 10-VM), at concentrations that inhibited incorporation of adenosine into cellular nucleotides by 85-90%, increased adenosine concentration (nmoles/107 cells) inside the cells from 0.05 ± 0.02 to 0.20 ± 0.09, 0.26 ± 0.13 and 0.20 ± 0.08, respectively (Table III). T h i s was accompanied by a reduction in adenosine concentration (nmoles/10 / cells) in the medium from 0.47 ± 0.15 to 0.Ii ± 0.03 in the presence of dipyridamole, 0.13 ± 0.05 in the presence of dilazep and 0.13 ± 0.04 in the presence of hexobendine (Table III). The effects of transport inhibitors, on inosine distribution followed a similar pattern as that described for adenosine (Table III). Incubation with AOPCP did not alter the concentration of adenosine in cells or media (Table III). Cells were 99.6 ± 0.1% intact at 0 min and 99.6% ± 0.1% at 30 min. In the additional presence of AOPCP, dipyridamole, dilazep or hexobendine cells were 99.5 ± 0.2%, 99.4 ± 0.1%, 99.4 ± 0.3% and 99.4 ± 0.1% intact, respectively. Discussion Nucleoside transport inhibitors, dipyridamole, hexobendine and dilazep inhibited incorporation of adenosine into cellular nucleotides in cultured chick embryonic heart cells with EC50 values which were comparable to those obtained previously (18) except in the case of dipyridamole which was ten times less potent in our study. The reasons for this difference is not known. Dipyridamole, hexobendine and dilazep at concentrations that inhibited incorporation of adenosine by 85 - 90%, increased accumulation of adenosine inside the cells from 10% to 60 - 90% and decreased efflux of adenosine by 70 90%, suggesting that adenosine is produced intracellularly and that its efflux occurs via the nucleoside transporter. Dipyridamole, dilazep and hexobendine
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Adenosine Formation and Release
Vol. 43, No. 23, 1988
also increased the amount of inosine in the cells from 13% to 98-99% and decreased that in the medium by 98-99%. This is consistent with inosine being exported out of the cell via the same transporter as adenosine (25). Nucleoside transport inhibitors have been shown to affect both release and uptake of adenosine in perfused hearts (9,26,27), in cultured neonatal rat heart cells (15) and freshly isolated hepatocytes (28). This is consistent with directionally symmetrical transport of adenosine (25,29). In contrast, Mustafa et al. (17), Mustafa (18) and Lee et al. (30) have reported the failure of dipyridamole, dilazep, hexobendine, lidoflazine and papaverine to inhibit adenosine release from embryonic chick heart cells. This conflict in results may be explained by the differences in methodology used to separate cells from medium. Some contamination of medium with cells would be inevitable with the method used by Mustafa et al, (17) and Mustafa (18) where incubations were terminated by scraping the cells from the culture dishes with a plastic spatula and separating them from the medium by centrifugation. In our hands this procedure resulted in 32.8 ± 4.7% cell breakage (n-7) compared to 0.39 ± 0.06% (n=7) when the medium was aspirated off and the cells separately extracted as in the present study. The method used by Mustafa et al. (17) and Mustafa (18) also resulted in leakage of adenine nucleotides into the medium thus exposing them to ecto-5'-nucleotidase and presumably there was also leakage of the accumulated intracellular nucleosides into the medium. An asymmetric effect of nucleoside inhibitors on release and uptake, however, has also been demonstrated in cultured aortic endothelial cells from pig (31) and cultured aortic smooth muscle cells from rat (32). Thus, although the possibilty of differences between cell types may exist, this does not appear to be the case between embryonic chick and neonate rat heart cells. In this study as in a previous study (15), nucleotides could not be detected in the medium. Furthermore, the concentrations of AMP in cells or medium were not altered by inhibition of the ecto-enzyme by AOPCP as would be expected if adenosine were being formed extracellularly or if the ecto- enzyme were acting as a translocase. In addition, inhibition of 5'-nucleotidase failed to prevent the production or release of adenosine from cultured chick embryonic heart cells in the present study, a finding consistent with other studies on neonatal rat heart cells (15), perfused hearts (9,33), isolated hepatocytes (28), brain slices (34), and rat polymorphonuclear leukocytes (35). Lee et al. (16), however, have reported that AOPCP eliminated the hypoxia induced release of adenosine in freshly isolated embryonic chick heart cells which resulted in extracellular accumulation of AMP. This discrepancy may be due to cell breakage that might be expected in freshly isolated cells. However, release of AMP and accumulation of AMP in the presence of AOPCP have also been demonstrated in perfused hearts (36). The present study demonstrates that adenosine formation in embryonic chick heart cells occurs via the same mechanism as described previously for neonatal rat heart cells (15) in which adenosine is formed intracellularly and is exported from the cell via the nucleoside transporter. Acknowledgements We gratefully acknowledge the travel grant received by Parviz Meghji from the Wellcome Trust in the U.K. and the expert technical assistance of Luvenia Wigginton and Mia Taylor. Supported by NIH grants HL-I0384 and HI-19242. References I. R. M. BERNE, Am. J. Physiol. 204, 317-322 (1963). 2. A.C. NEWBY, Trends Biochem. Sci. 9, 42-44 (1984). 3. P. HEDQVlST and B.B. FREDHOLM, Acta Physiol. Scand. 105, 120-122 (1979).
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4. 5. 6 7. 8. 9. I0. Ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
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