SHORT COMMUNICATION
Uptake and release of adenosine by cultured astrocytoma cells (Rrceiued 14 November 1978. Acwpri,d 6 February 1979)
containing 5 ml of culture medium, and incubation was ADENINF derivatives have been implicated as neurotransmitters in the autonomic nervous system (BURNSTOCK, carried out for 60min. The medium was then aspirated 1972, 1975), and several observations have suggested that and the cells were washed twice with the incubation fluid adenosine may in some way be involved in synaptic described by PULL & MCILWAIN (1972). Release experitransmission in the central nervous system as well. Adeno- ments were carried out in the same medium for 20min, sine is released from brain slices incubated in uitro (PULL and incubations were terminated by aspirating the & MCILWAIN, 1972), and this release is augmented by elec- medium. & MC~LWAIN, 1972) and by depotrical stimulation (PULL Sepuration of I4C derivatives. The aspirated medium was larizing agents (SHiMizu rt a/., 1970h; LEwiN & BLECK, brought to a volume of lOml with 0.1 N-HCI. A 1 ml ali1976). Brain slices also take up adenosine from the incuba- quot was removed for counting, and the liberated comtion medium (SHiMizu et ul., 1969). In addition, adenosine is pounds contained in the remaining 9 ml were concentrated & MCILWAIN, by charcoal adsorption, eluted, and separated by thin layer liberated from synaptosomal beds (KURODA 1974) and from the surface of rat cortex following electrical chromatography as described previously ( L ~ W I& N BLECK, & PHILLIS, 1975). When adenosine stimulation (SULAKHE 1976) using the solvent mixture of SHIMIZUet crl. (1969). I S applied iontophoretically to cortical neurons, inhibition Uptake of [14C]adenosinr. For uptake experiments the of discharge has been most frequently observed (PHILLIS growth medium was aspirated, and the cells were washed t’i cil., 1974). Adenosine stimulates cyclic A M P accumutwice with incubation fluid. Two milliliters of the same lation in brain slices (SATTIN& RALL,1970), and the phymedium containing the desired concentration of siologic action of adenosine may be mediated by cyclic [14C]adenosine (100, 10, 5, 2, I , 0.5, 0.2. or 0.1 pM) was AMP. then added and incubations were carried out for 10min The contribution of glia to the observed release of (uptake with time being linear for at least 20min). The adenosine from brain slices is unknown. Cultured astrocymedium was then aspirated, and the cells were washed toma cells offer a population of cells of glial origin. twice with medium and then extracted with 1 ml of 57” Although tumor cells may be poor models of normal adult trichloroacetic acid. The extract was diluted to a bolume glia in civo, we elected to study the release as well as the of 10ml with 0.1 N-HCI. A 1 ml aliquot was removed for uptake of adenosine in cells derived from tumor astrocytes. counting, and the adenine derivatives in the remaining 9 ml were concentrated and separated as previously described for adenine derivatives (LEwiN & BLECK,1976). MATERIALS AND METHODS Mafericrls. [‘“C]Adenosine (53 mCi/mmol) and [ “ C l adenine (50 mCi/mmol) were purchased from SchwartzMann, Orangeburg, NY. Eagle’s minimal essential medium wdS supplied by Flow Laboratories, Inglewood, CA. Fetal calf serum was from Microbiological Associates, Los Angeles. CA. Theophylline and nonradioactive nucleotides nucleosides, and purine bases were purchased from Sigma, St. Louis, MO, and dipyridamole was provided by Boehringer Ingelheim. Elmsford, NY. Scintillation fluid (0.56% Omnifluor) was supplied by New England Nuclear, Los Angeles, CA. Cell cultures. The astrocytoma cells used in these experiand were ments were obtained from Dr. JOHN PERKINS subclone (1321N1) of a line (1181N1) derived from human tumor astrocytes. The experiments were performed on cells grown in Falcon plastic petri dishes in 5 ml of Eagle’s minimal essential medium with added glutamine, penicillin and streptomycin, and supplemented with 10% fetal calf serum under an atmosphere of air + C 0 2 (95:5) at 37°C in a humidified incubator. Dishes were seeded with approx 3 x lo5 cells and grown for 5 days. Cultures grown for this period had not become confluent. Release qf[14C]adenine derivatives. In order to prelabel the cells, 1 pCi of [I4C]adenine was added to each dish
RESULTS The effluent from cultured glial cells prelabeled with [‘4C]adenine was composed largely of inosine (73u9 and hypoxanthine (23”/,), with small amounts of adenosine and of other adenine derivatives (Table 1). Adenosine (0.1 mM) produced a 28”,;, increase in the release of ‘‘C-labeled compounds ( P < 0.05). The results of thin layer chromatography suggested that the increase consisted largely of inosine in addition to a small increase in adenosine. 2-Chloroadenosine had no effect on ‘“C efflux. The enhanced release evoked by adenosine was completely blocked by dipyridamole (0.1 mM) but was not inhibited by theophylline (0.5 mM). Double reciprocal plots of adenosine uptake after 10 min incubations are presented in Fig. 1. Both the uncorrected data and the results obtained by subtracting values obtained at 0°C are shown. For the uncorrected results, ~ ~ V,,.,, 0.14pmol x g prothe apparent K , was 1 . 0 4 and tein-’ x m%’. After correction, K , was 0.95 p~ and V,,,, 0.11 pmol x g protein-’ x min-’. At M-adenosine almost all of the recovered ’“C was contained in nucleotides. As the concentration of adenosine increased, the proportion of ‘“C present as inosine and adenosine also in-
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T A B L1.~ [14C]ADENINE
DERIVATIVES
RELEASED FROM CULTIIRFI) CELLS
Control
ASTROCYTOMA
Adenosine (0.1 mM)
2-Chloroadcnosinc (0.1 mM)
Total
81.2
* 3.77
104 k 3.32
76.3 2 4.17
Adenosine Inosine Hypoxanthine Other Total activity recovered Recovery (%)
0.44 +. 0.11 32.6 i-1.86 10.1 f 0.66 1.71 f 0.31
1.20 2 0.52 51.8 _+ 0.66 8.41 0.65 1.71 k 0.42
0.58 k 0.28 26.4 k 2.05 10.6 2 0.54 2.00 0.54
44.9 55.2
63. I 60.7
39.6 51.9
*
Each value is the mean
Activity expressed as nCi/mg tissue protein of four experiments. creased so that at M-adenosine, 23% of the recovered radioactivity was in inosine and 9% in adenosine with 65% remaining in nucleotides. The adenine nucleotides were not further separated. DISCUSSION After prelabeling with [Wladenine, the efflux from cultured glioma cells incubated in uitro consisted largely of inosine and hypoxanthine, the immediate metabolites of adenosine, with only a small amount of adenosine in the effluent. This finding is consistent with the presence of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) activity in cultured glia (TRAMS & LAUTER,1975). The addition of adenosine to the medium significantly enhanced the release of radioactivity, largely accounted for by an increase in inosine. It seemed likely that the effect of adenosine either resulted from its uptake and displacement of intracellular adenosine, which was then deaminated, or from its interaction with a membrane receptor. The augmentation of efflux of radioactivity was blocked by dipyridamole, an inhibitor of adenosine uptake (KOLASSAet ul., 1970; CLARKet al., 1974), but was not altered by theophylline. In contrast, CLARKet a/. (1974) found that the stimulation of cyclic A M P accumulation by adenosine in this cell line was inhibited by theophylline
S.E.M.
*
but not by dipyridamole. observations thought to indicate that adenosine was acting extracellularly on a membrane receptor. Moreover, 2-chloroadenosine, an analogue which markedly stimulates cyclic A M P formation in brain slices (HUANGrt ul., 1972). did not increase the efflux of labeled compounds. This analogue is poorly incorporated into nucleotides by cultured brain cells (STUROILLet d.,1975) and would not be expected to displace adenosine. These findings suggest that the enhanced release of [I4C]adenine derivatives produced by adenosine was dependent on its uptake rather than o n an extracellular action. Studies of adenosine uptake demonstrated a high affinity incorporation. Adenosine uptake by this cell line (CLARK et ul., 1974) and by C-6 rat glioma cells (SCHULTZ et ul., 1972) has been observed, but kinetic data have not been reported. The observed kinetic constants are in the same range as those recently found by HERTZ(1978) in astrocytes cultured from newborn mice. At lo-’ M-adenosine almost all recovered radioactivity was contained in adenine et a/. (1969) found in brain nucleotides. Similarly, SHIMIZU slices that a low concentration of adenosine was entirely incorporated into nucleotides. At high adenosine concentrations, an increasing proportion of radioactivity was present as inosine and adenosine, resembling the observa& DEAN(1977) in erythrocytes. Unlike tions of PERRETT adenosine neither inosine nor hypoxanthine stimulates cyc-
._ E
0
5
10
VADENOSINE (pM-’)
FIG.1. Reciprocal plots of [‘4C]adenosine uptake in cultured astrocytoma cells as a function of reciprocal adenosine concentrations (0.1-100 PM). Kinetic constants were determined by the method of least squares. Uncorrected data plotted as open circles. Data from which values obtained at 0°C have been subtracted shown as solid circles.
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rivatives from cerebral cortical slices: effect of phenytoin and phenobarbital. Neurochem. Res. 1, 429-435. PERRETTD. & DEANB. (1977) The function of adenosine deaminase in the human erythrocyte. Biochem. biophys. Res. Commun. 77, 374-378. PHILLIS J. W., KOSTOPOULOS G. K. & LIMACHER J. J. (1974) Depression of cortico-spinal cells by various purines and pyrimidines. Can. J . Physiol. Pharnzuc. 52, 1226- 1229. PULL I. & MCILWAIN H. (1972) Metabolism of [14C]adenine and derivatives by cerebral tissues, superNeurology Service, Veterans EDWARDLEWIN fused and electrically stimulated. Biochem. J . 126, Administration Medical Center, VIRGINIA BLECK 965-973. and the, Depurtnzeiit of Neurology, SATTINA. & RALLT. W. (1970) The effect of adenosine Uniivrsitj, of Colorado Medical Center, and adenine nucleotides o n the cyclic adenosine Dunvur, CO, U.S.A. 3'3-phosphate content of guinea-pig cerebral cortex slices. Molec. Pharmuc. 6, 1 S 2 3 . J., HAMPRECHT B. & DALYJ. W. (1972) AccumuSCHULTZ lation of adenosine 3': 5'-cyclic monophosphate in clonal glial cells: labeling of intracellular adenine nucleotides REFERENCES with radioactive adenine. Proc. nutn. Acad. Sci.. U . S . A . G. (1972) Purinergic nerves. Pharmuc. Reu. 24, BURNSTOCK 69, 1266 1270. C. R. (1969) A SHIMIZUH., DALYJ. W. & CREVELING 509-58 1, radioisotopic method for measuring the formation of BURNSTOCK G. (1975) Purinergic transmission, in Handadenosine 3',5'-cyclic monophosphate in incubated slices hook of Psychophormacology (IVERSENL. L., IVERSEN s. of brain. J . Neurochem. 16, 1609-1619. D. & SNYDER S. H.. eds.) Vol. 5, pp. 131--194. Plenum H., CREVELINC C. R. & DALYJ. W. (1970ci) Effect SHIMIZU Press. New York. CLARKR. B., GROSSR., SU Y.-F. & PERKINS J. P. (1974) of membrane depolarization and biogenic amines on the formation of cyclic AMP in incubated brain slices, in Regulation of adenosine 3': 5'-monophosphate content in Role of Cyclic A M P in Cell Function, Aduances in Biohuman astrocytoma cells by adenosine and the adenine P. & COSTA chemical Psychopharmacology (GREENCARD nucleotides. J . biol. Chem. 249, 5296-5303. E., eds.) Vol. 3, pp. 135-154. Raven Press, New York. HERTZL. (1978) Kinetics of adenosine uptake into astroSHIMIZU H., CREVELING C. R. & DALV J. (1970h) Stimucytes. J . Neurochem. 31, 55-62. lated formation of adenosine 3',5'-cyclic phosphate in HUANGM., SHIMIZUH. & DALYJ. W. (1972) Accumulation of cyclic adenosine monophosphate in incubated cerebral cortex: synergism between electrical activity and slices of brain tissue. 2. Effects of depolarizing agents, biogenic amines. Proc. natn. Accid. Sci., U . S . A . 65, membrane stabilizers, phosphodiesterase inhibitors, and 1033-1040. STURGILL T. W.. SCHRIERB. K. & GILMANA. G. (1975) adenosine analogs. J . men. Chem. 15, 462-466. W. (1970) Specificity KOLASSAN., PFLEGERK. & RUMMEL Stimulation of cyclic AMP accumulation by 2-chloroof adenosine uptake into the heart and inhibition by adenosine: lack of incorporation of nucleoside into cycdipyridamole. Eur. J . Pharmuc. 9, 265-268. lic nucleotides. J . Cyclic Nucleotide Res. 1, 21-30. KURODAY. & MCILWAINH. (1974) Uptake and release SULAKHEP. V. & PHILLISJ. W. (1975) The release or of ['4C]adenine derivatives at beds of mammalian corti3H-adenosine and its derivatives from cat sensorimotor cal synaptosomes in a superfusion system. J . Neurochem. cortex. Life Sci. 17, 551-556. 22, 691-699. TRAMS E. G. & LAUTERC. J. (1975) Adenosine deaminase of cultured brain cells. Biochem. J . 152, 681 687. LEWlN E. & BLECKV. (1976) Release of I4C-adenine de-
lic AMP accumulation in brain slices (SATTIN & RALL, 1970; SHIMIZUet a/., 1970a), and their physiologic action is unclear. If astrocytes in uiuo are similarly able to take up and to metabolize adenosine either by incorporation into nucleotides or by conversion to inosine and hypoxanthine, which are then released, these cells might play a role in inactivating this compound.