[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

.Neurochemistry International. Vol. 5, No. 2, pp. 245 to 249, 1983 Printed in Great Britain

0197-0186/83/020245-05503.00/0 © 1983 Pergamon Press Ltd.

[3H]ADENOSINE BINDING SITES ON 108CC15 NEUROBLASTOMA x GLIOMA HYBRID CELL LINE A N D RAT BRAIN MEMBRANES PENELOPE H. SNELL a n d CHRISTOPHER R. SNELL MRC Neuroendocrinology Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, U.K. (Received 24 August 1982; accepted 1 October 1982) Al~traet--Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [3H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, Kds 1.4 and 29.1 pM and a single low affinity site on the hybrid cells, Kd 2.6/tM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an 'R' adenosine receptor of the A2 subtype. It is concluded that [3H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.

Adenosine, in addition to its role in intermediary metabolism is t h o u g h t to function in the central nervous system as a neurotransmitter or n e u r o m o d u l a t o r (Burnstock, 1980; Stone, 1981). Purinergic binding sites have been demonstrated on brain membranes using the following ligands; [3H]2-chloroadenosine (Wu eta[., 1980; Williams and Risley, 1980). [3H]N6cyclohexyladenosine (Bruns et al., 1980), [ 3 n ] N 6 phenylisopropyl adenosine (PIA) (Schwabe and Trost, 1980) and [3H]adenosine (Schwabe et al., 1979; N e w m a n et al., 1981). In those studies that used adenosine as labelled ligand, two low affinity binding sites were revealed, whereas the other labelled ligands showed binding sites of high affinity. At micromolar concentrations adenosine can stimulate c A M P production in brain slices (Daly, 1977), glial cells (Van Calker et al., 1979), neuroblastoma cells (Blume et al., 1973) and n e u r o b l a s t o m a × g l i o m a 108CC15 hybrid cells (Sharma et al., 1975) and at submicromolar concentrations can inhibit c A M P production in cultured glial cells (Van Calker et al., 1979). In this paper, we investigate the [3H]adenosine binding sites on 108CC15 neuroblastoma x glioma hybrid cells and compare them with those present on brain membranes.

of Dr B. Hamprecht, Wurzburg, FRG. The cells were grown in 75 cm 2 flasks at 37°C in Dulbecco's modified Eagle's medium containing 10~ foetal calf serum, 100 ,uM hypoxanthine, 1/IM aminopterin and 16 #M thymidine in a humidified atmosphere of 10~o CO2-90~o air. Cells with passage number between 14 and 20 were harvested at 80~ confluency (approx. 6 x 106 cells/flask), washed twice with washing buffer (137mM NaC1; 5.4raM KCI; 0.17mM Na2HPO4, 0.22 mM KH2PO4, 5.5 mM glucose) and resuspended in assay buffer (50 mM Tris, 0.1~o BSA; pH 7.4) at a concentration of 106cells/ml for use in the binding assays. Cells were counted using a haemocytometer. Brain membrane preparation Rat brains without cerebellum were rapidly removed and homogenised in 10 vol. of ice cold 0.32 M sucrose. After centrifugation at 1000g for 10min to remove the nuclear fraction and cell debris, the supernatant was centrifuged at 45,000 g for 45 min. This crude synaptosomal fraction was used by rehomogenisation in 0.01 M Tris, pH 7.4, and centrifuged at 45,000 g for 30 min. Membranes were washed three times by rehomogenisation in 0.01 M Tris, pH 7.4 and subsequent centrifugation. The final pellet was suspended in buffer (5 ml/g original tissue) and frozen in aliquots at -70°C for use in binding assays. The protein concentration was determined by the method of Lowry et at. (1951). Receptor binding assays High affinity site by dilution of label. Lysed synaptosomal membranes (0.5mg protein) or 108CC15 cells (5 x l0 s cells) were added to assay tubes containing [2,5',8-3H]ade nosine (Amersham International Limited, 42Ci/mmol 0.5-20 nM) in a final volume of 1 ml of buffer (0.05 M Tris, 0.1~o BSA, pH 7.4). A parallel series of tubes were incubated in the presence of 5 x 10-4M adenosine or 1 x 10 5M PIA. After 30min at I°C the tubes were cen-

EXPERIMENTAL PROCEDURES Cell culture and harvest 108CC15 neuroblastoma x glioma hybrid cells were obtained from Dr B. Gustelson with the kind permission 245

246

PINtlC+I,I H. ~NI:I.[ arid ( IIRIS'I(H'tn:R R. SNtl I

trifuged at 14,000,q for 3 min to separate botmd from free label. The supernatants were aspirated off and the pellets superficially washed with ice cold buffer {0.2 mb. The tips of the tubes were cut off into scintillation vials and each pellet suspended in water (0.5 ml). After addition of Puckard 299 scintillation (4 ml) the radioactivity in the pellet was measured on a Packard 460CD scintillation counter with on line dpm correction. All binding assays were performed in triplicate. The specific binding ~as taken as the difference between the binding in the absence and presence of excess cold ligand. Low affinity sites hy displacement ,stud),. 108CC15 cells ~5 × 10s cells/tubes) or synaptosomal membranes (0.5 mg protein) were maintained at I C for 15 min with 3H-adenosine (I.6 pmol/tube) before addition to a serial dilution of unlabelled adenosine or adenosine analog in a linal volume of 1 ml. After a further 30 rain at 1 C the bound ligand was separated from free by centrifugation at 14,0000 for 3 rain and the radioactivity in the pellet measured as for the high affinity site. Data analysis. The high affinity and low affinity binding data were analysed by the method of Scatchard (1949). Contributions from multiple sites were separated mathematically from the Scatchard plot (Burr and Snyder. 1975). RESULTS

[SH]Adenosine binding to 108CC15 neuroblastoma × ,qlioma hybrid cells Scatchard analysis of the high affinity binding site for [3H]adenosine on 108CC15 neuroblastoma x glioma hybrid cells is shown in Fig. 1. Mathematical extraction of the contributions from the two components using the method described by Burt and Snyder (1975) allowed the high affinity site to be quantitated, see Table 1, The displacement curve for the low affinity binding site on these cells. Fig. 2, was analysed by the method of Scatchard and showed a single binding site in the micromolar range with K d and Bmax as shown in Table l. The density of the low and high affinity binding sites varied with the growth of the cells. The densities were at their lowest when the cells were overgrown or just after splitting, nevertheless if cells were harvested

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0,4

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.

.

.

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14

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Fig. 1. Scatchard plot of high affinity binding of [~H]adenosine (0.5 20nM) to 108CC15 neuroblastoma x glioma hybrid cells. between 60 and 80% confluency the densities remained acceptably reproducible. When PIA was used in place of adenosine for determination of nonspecific binding in the high affinity experiments the receptor density of the high affinity site was unchanged at 36fmol/106 cells but the affinity was increased two fold over adenosine to 0.47 x 10 -9 M. Table 2 shows the ICso values of a range of adenosine related c o m p o u n d s for the [3H]adenosine labelled low affinity binding site on 108CC15 cells. The ICs0 values represent the concentration of analog required to displace 50% of the specific [3H]adenosine binding.

[3H]Adenosine binding to lysed synaptosomal membranes Figure 3 shows the Scatchard analysis of the high affinity binding of [SH]adenosine to lysed synaptosoreal membranes obtained using procedure described in "Experimental Procedures'. Two distinct binding sites were apparent and the contribution from the two sites obtained by mathematical analysis. The K~ and Bm,, of the high affinity site are shown in Table 1.

Table 1. Dissociation constants, Kd, and receptor densities, B .... of [3H]adenosine binding to 108CC15 cells and brain membranes using adenosine as displacing agent 108CC15 cells

Brain membranes Bmax

Bmax

KL~ High affinity site Low affinity site I Low affinity 2

0.86 _+ 0.t5 nM 2.62 ± 0,38 ,uM

(fmol/10~' cellsl 43.4 ± 10,9 154,400 ± 39.000

Ka 3.1 + 1 nM 1.41 _+ 0.6 ~LM 29.1 ± 9.9 ,uM

(fmol/mg protein) 17.2 _+ 4.6 6040 _+ 1400 144,000 ± 28,00

High affinity and low affinity binding obtained and analysed as described in "Experimental Procedures'.

Jail]Adenosine binding to 108CC15 cells and CNS membranes 100

....-4

247

Table 2. ICso values, of adenosine analogs required to displace 50~o of specific [3H]adenosine binding to the low affinity site on 108CC15 hybrid cells ICso (/~M)

i

i ° 10-7

~-¢

1Cr-6

[ JldlldlOlli~]

10-4

Adenosine 6-Methyladenosine PIA 2'Deoxyadenosine YDeoxyadenosine cAMP 2'5'Adenyladenosine Adenosine triphosphate Adenine

2.5 4.6 3.1 14.3 > 100 > 300 > 300 > 300 > 300

(M)

Fig. 2. Low affinity binding of [3H]adenosine (1.6 nM) to 108CC15 hybrid cells obtained by displacement of specific [3HJadenosine binding by cold adenosine as described in Methods. The low affinity binding of [3H]adenosine to synaptosomal membranes obtained by displacement of r3H]adenosine binding with increasing concentrations of adenosine was analysed by the method of Scatchard as in Fig. 4. Two low affinity sites were found and the dissociation constants and receptor densities of the two components are shown in Table 1. The high affinity receptor was found to be lost after repeated washing of the membranes (greater than 5 times), after preincubation of the membranes at 20°C for 30 min followed by replacement of the medium; and after storage overnight at 4°C.

IC50 values obtained from displacement studies as described in 'Experimental Procedures'. ATP, whereas at the P2 receptor the reverse is true. Londos and Wolff (1977) have studied the structureactivity requirements of purine susceptible adenylate cyclase systems in a variety of tissues and identified two receptor subtypes which they termed 'R' and 'P' sites. The 'R' site is extracellular, is associated with an increase in cAMP levels and requires an intact ribose portion of the molecule for activity, whereas the 'P' site is intracellular, very sensitive to changes in the purine region of the nucleotide and involves an inhibition of cAMP production. Using culture glial cells Van Calker et al. (1979) have further subdivided the 'R' site into A1 and A 2 receptors. The A~ sites are of

DISCUSSION Pharmacological studies by Burnstock (1980) have demonstrated Pa and P2 purinergic receptor types. At the P1 receptor adenosine is more potent than 10

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K

Bound (pmol/O-Smg protein) 4 50

100

150

Bound (fmo4/O'Srngproton)

Fig. 3. Scatchard plot of high affinity binding of [3H]adenosine (0.5 20 nM) to brain synaptosomal membranes.

Fig. 4. Scatchard plot of low affinity binding sites for [3H]adenosine (1.6nM) on brain synaptosomal membranes obtained by displacement of specific [3H]adenosine binding by cold adenosine as described in 'Experimental Procedures'.

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high affinity and associated with an inhibition of c A M P levels, whereas the A 2 sites are of lower affinity and inw~lve an increase in c A M P levels. The A1 and Ae receptors tire stimulated preferentially by PIA and adenosine, respectively. Londos et al. (1980) have proposed a similar subdivision of the 'R" site into an inhibitory, Ri, and a stimulatory. Ra, component based on the effects of receptor stimulation on adenylate cyclase activity. Ligand binding studies on brain membranes using [3H]adenosine, have revealed two binding sites with dissociation constants in the micromolar range (Schwabe et al., 1979; Newman et al., 1981), whereas [3H]PIA, [3H]2-chloroadenosine or [-tH]N~'-cyclohexyladenosine appear to bind to one or two sites with nanomolar affinity (Wu et al., 1980: Williams and Risley, 1980: Bruns et al.. 1980: Schwabe and Trost, 1981). These differences in ligand affinity could reflect PIA, 2-chloroadenosine and N~-cyclohexyladenosine binding with high affinity to the lower affinity [3H]adenosine binding sites or these ligands could be binding with high affinity to sites distinct from those already described for [3HIadenosine. Such binding data is difficult to correlate with the pharmacological studies on adenosine receptor subtypes previously reported. In the present study we have been able to demonstrate the two low affinity adenosine binding sites on brain membranes reported by Schwabe et al. (1979) and Newman et ul. (1981) with affinities in good agreement with their data. Most significantly, we have shown the presence of a high affinity adenosine binding site on brain membranes using [3H]adenosine as labelled ligand. Previous workers who have also used [3H]adenosine have not detected this binding site probably due to the presence of endogenous adenosine in the membrane preparations and the lability of the high affinity receptor. We have found that using carefully washed membranes and incubating at I ' C the activity of the adenosine synthesizing enzymes is reduced considerably, thus allowing reliable and reproducible measurement of the high affinity site. The production of c A M P in 108CC15 neuroblastoma x glioma hybrid cells can be stimulated by a variety of neurochemical agents including adenosine (Sharma et al., 1975). As the pharmacological subclassification of adenosine receptors is reliant on the effects of receptor stimulation on c A M P production, this cell line is an ideal system to correlate binding studies with effects on adenylate cyclase. We have shown the cell line to possess the high affinity binding site present on rat brain membranes, but the cell line possesses only the higher of the two micromolar affinity adenosine binding sites found on brain mem-

branes. The receptors we have found on these cells are necesarity extracellular because the experiments are conducted on whole cell preparations. In addition the contribution from adenosine uptake to the observed binding is minimal because the assays are performed at I C in the absence of sodium ions: conditions which severely inhibit the uptake process for adenosine (Bender et al., 1980; Barberis el al., 19811. The low affinity binding site otl the hybrid cells was investigated for its structural requirements for ligand affinity, see Table 2. Analogs involving modification to the purine region of adenosine appeared to retain affinity for the binding site comparable with the parent molecule. However. analogs with modification to the ribose ring were much less able to displace [3H]adenosine from this binding site. Clearly the micromolar binding site on the hybrid cells is of the 'R" type described by Londos & Wolff (1977). The concentrations of adenosine required to stimulate cAMP production in 108CC15 hybrid cells is comparable with the dissociation constant of this binding site (Hamprecht, 1977). The subctassification by Van Calker et al. (1979) of the "R' site into A1 and A2 sites was established using cultured gtial cells; in their systems PIA was more potent than adenosine at the A~ site whereas at the A2 site the reverse was true. We would deduce, therefore, that the micromolar binding site on the 108CC15 hybrid cells is of the A2 subtype as it is associated with c A M P stimulation and PIA is not significantly more potent than adenosine at this site. Structure activity studies on the high affinity site were not possible using displacement methods due to the relatively large contribution of the low affinity site to the overall binding. However. it could be speculated that as PIA more readily displaces [3H]adenosine in the high affinity experiments that this site could be an "R' binding site of the A~ subtype. Further work on cAMP production in these cells should clarify this possibility. It is likely that the high affinity sites demonstrated with [~H]PIA, [3H]chloroadenosine and [3H]cyclohexyladenosine corresponds to this high affinity [3H]adenosine site and not the lower affinity sites. The observation that the density of adenosine receptors varies with cell growth has particular relevance as adenosine receptors have been strongly implicated in cell growth inhibition (Henderson and Scott, t980). In particular, cell division in 108CC15 hybrid cells can be inhibited by culture in the presence of 1 mM dibutyryl c A M P (Nelson et ul., 1976; Reiser et al.. 1977), concomitantly the cells become more differentiated and extend neurites from the cell

[3H-[Adenosine binding to 108CC15 cells and CNS membranes body. The observed receptor densities are lowest when the cells are growing at low density, e.g. after splitting, and highest when the monolayer becomes confluent. It could be speculated that adenosine receptor levels are elevated when the cells are growing fast and the monolayer is becoming crowded, and when stimulated these receptors function to inhibit cell growth at a time when cell survival is threatened. W h e n the cells are growing freely at low cell density, growth inhibition is not appropriate and the receptor density is low. We conclude that [3H]adenosine can be used as a labelled ligand to demonstrate b o t h high and low affinity binding sites on brain m e m b r a n e a n d 108CC15 hybrid cells, and that the 108CC15 cells possess only one low affinity adenosine receptor that is of the A2 subtype. The 108CC15 hybrid cells represent a valuable model for studying the biochemistry and neurochemistry of the 'R' type adenosine receptors.

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