Solubilization of an Adenosine Uptake Site in Brain

Abstract: Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine ([3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 ± 0.02 nM and 133 ± 18 fmol/mg protein, respectively. with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep > hexobendine > dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine ([3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high‐affinity site with a KD of 0.32 ± 0.06 nM and a Bmax of 105 ± 30 fmol/mg protein and a loweraffinity site with a KD of 5.50 ± 0.52 nM and Bmax of 300 ± 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake ([3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.

sine receptor ligands are much less potent inhibitors of binding, and caffeine i s without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake ([3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state. Key Words: Solubilized adenosine uptake sites-Nitrobenzylthioinosine-Adenosine receptors. Verrna A. et al. Solubilization of an adenosine uptake site in brain. J . N~itrochern. 45, 596-603 (1985).
Adenosine is released in a depolarization-induced, calcium-dependent manner (Stone,198 I ) and its effects are thought to be mediated by specific, high-affinity, cell-surface recognition sites that have been described in brain using radiolabeled stable adenosine analogs (Bruns et al., 1980. Termination of adenosine action is thought to be regulated primarily by a facilitated reuptake mechanism involving specific elements in the cell membrane (Paterson, 1979;Bender et al., 1980;Barberis et al., 1981). The activity of this adenosine uptake mechanism is important since agents that inhibit this process have been shown to potentiate the neuromodulatory effects of adenosine (Huang and Daly, 1974;Phillis et al.. 1979;Crawley et al., 1983; Phillis and Wu, 1 9 8 3~) .
Inhibition of adenosine uptake in peripheral tissue is thought to be the mechanism of action of vasoactive drugs such as dilazep and dipyridamole (Sano, 1974). Such an inhibition may also be partially responsible for the actions of centrally active drugs such as benzodiazepines and phenothiazines (Phillis and Wu, 19830).
Recent investigations examining adenosine uptake sites directly have employed binding studies using [3H]nitrobenzylthioinosine ([3H]NBI), which is a potent, selective inhibitor of adenosine uptake into both nervous (Barberis et al., 1981) and nonnervous tissue (Pickard and Paterson, 1972;Brajeswar et al., 1975). [3H]NBI has been shown to bind specifically, reversibly, and with high affinity to adenosine uptake sites in erythrocytes (Jarvis and Young, 1980), HeLa cells (Lauzon and Paterson, 1977), and brain (Marangos et al., 1982). Hammond et al., in studies using human erythrocytes (Hammond et al., 1982) and guinea pig cortical tissue (Hammond andClanachan, 1982, 1983), have shown that displacement of [3H]NBI binding by a test compound was indicative of that compound's ability to block adenosine uptake into these tissues. Thus, the measurement of site-specific binding of [3H]NB1 seems t o offer a convenient probe for studying molecular interactions at adenosine uptake sites just a s the stable radioactive a d e n o s i n e analogs d o for adenosine receptors.
O n e of the fundamental approaches in understanding t h e biochemistry of t h e s e membranebound sites is their solubilization in a functional state. Several recent studies have used this a pproach t o examine adenosine receptors in brain (Gavish e t al., 1982;Bruns et al., 1983; Nakata a n d Fujisawa, 1983). Using [3H]NBI a n d cycloh e~y l [~H ] a d e n o s i n e ([3H]CHA) as probes, we now report successful solubilization of both adenosine uptake sites and receptors from guinea pig brains.
[3H]NBI-labeled uptake sites are characterized and are seen t o retain pharmacological and binding kinetic properties similar t o those described in membrane bound preparations.

Membrane preparation
Frozen rat or guinea pig whole brains were suspended in 10 volumes of cold 50 mM Tris-HCI buffer (pH 7.5) containing 10 mM EDTA, 100 M phenylmethysulfonyl fluoride (PMSF), and 1 rnM ],lo-phenathroline using a Brinkman Polytron (speed 5 for 30 s). The suspension was centrifuged at 30,000 g for 20 min and the resulting pellets were suspended in 10 volumes of 50 rnM Tris-HCI (pH 7.5) and spun again as above. The following pellets were resuspended in Tris buffer and incubated with 2 Uiml of adenosine deaminase at room temperature for 30 min. The suspensions were centrifuged again and the final pellets wcrc stored at -70°C. Pellets thus stored were found to be stable for several weeks.

Detergent solubilization
Solubilization of membrane pellets was accomplished using the method described by Bruns et al. for solubilizing various receptors (Bruns et al., 1983). Briefly, 1 g (tissue wet weight) of membrane pellet was thawed and suspended in 20 volumes of 50 mM Tris-HCI and centrifuged at 30,000 g for 20 min. Using a pasteur pipet, the washed pellet was finely resuspended in 2 ml of solubilization buffer containing 10 mM Tris-HCI, 1 mM dithiothreitol (DTT), and either 20% glycerol or no glycerol at 0°C. TWO milliliters of solubilization buffer with I% CHAPS deter-gent was added, and the suspension was swirled gently for 30 s and centrifuged at 145,000 g for 1 h in a Beckman L8-55 ultracentrifuge using a 45-Ti rotor. The supernatant was removed with a pasteur pipet and passed through a Millipore 0.22 p.M filter. The filtrate was either assayed directly for binding or stored at -20°C. To test the effect of glycerol on stability of the solubilized extracts, the preparation extracted without any glycerol was adjusted to contain either 5 or 20% glycerol. Subsequently, all binding experiments were done with supernatant that had been adjusted to contain 20% glycerol after the extraction.
[ 3H]NBI binding assay Solubilized extracts (0.3-0.4 rng of protein) were incubated in the presence of [3H]NBI (17 Ci/mmol. Moravek Biochem) in 50 mM Tris-HCI buffer (pH 7.4) (final volume of 0.5 ml) for 30 min at 22°C. Assays were terminated by rapid vacuum filtration through Whatman GF-B filters that had been presoaked for 1-24 h in 0.3% polyethylenimine (,Sigma). The filters were subsequently washed quickly with 4 x 3 ml washes of ice-cold Tris buffer. Filters were air-dried and counted in 10 ml of Redi-Solv scintillation fluid (Beckman). Nonspecific binding was determined by adding 5 p,M NBI (Calbiochern) in the assay and routinely represented 10% or less of the total binding at 0.7 nM t3H]NBI. Specific binding was determined by subtracting the nonspecific binding values from total binding values. Scatchard analyses were performed by incubating the soluble extracts with [3H]NBI concentrations ranging from 0.05 to 2.5 nM. Inhibition studies were done by incubating the extracts in the presence of six different concentrations of inhibitor and 0.7 nM ['HINBI. All binding data are expressed as specific binding and all assays were performed in triplicate. K, values were calculated using the relationship K , [3H JCHA binding assay [3H]CHA binding to soluble extracts was measured in a manner similar to that described above for [3H]NBI binding, except that the incubation was performed at 22°C for 2 h. Nonspecific binding was determined by incorporating 50 K M CHA (Calbiochem). Scatchard analysis was performed by incubating extracts with [3H]CHA (25 Ci/rnmoI) concentrations ranging from 0.40 to 25.0 nM. Inhibition studies were done using 5 nM [3H]CHA. For calculating K , values the K , value for [3H]CHA binding was taken as 1 nM rather than using the component highand low-affinity K , values. All protein determinations were done using the Biorad protein assay.
Veraparnil was a gift of Knoll Pharmaceuticals and nimodipine of Miles Pharmaceuticals. In experiments utilizing nimodipine, procedures were performed in darkened rooms. Dilazep was a gift of Hoffmann La Roche (Nutley), CHA was obtained from Calbiochem, and L-PIA and all other chemicals used were obtained from Sigma.

Binding characteristics
The site-specific binding of ['HINBI to soluble extractions from guinea pig and rat brains was proportional t o the protein concentration in the assay over the range of 0.04-0.60 rng protein/ml as shown in Fig. 1. Saturation of binding was achieved above a protein concentration of 0.60 mg/ml. In all experiments performed. final protein concentrations ranged between 0.30 and 0.40 mg/ml. Figure 2 shows the effect of glycerol in stabilizing the activity of solubilized guinea pig extractions. Preparations extracted with 20%' glycerol in the solubilization buffer and those that were reconstituted with 20% glycerol after extraction in glycerol-free buffer showed no loss of specific binding activity over a 2-week period. Reconstituting glycerol-free extractions with 5% glycerol led to a loss of about 30% of the initial specific binding after a 2-week period. In the absence of glycerol, preparations were found to retain only 50% of the initial specific binding after 1 week and after 2 weeks almost 90% of the activity was lost. Preparations extracted with 20% glycerol included in the solubilization buffer were cloudy or translucent in appearance, even after filtration through 0 . 2 2 -~m filters whereas the preparations to which glycerol was added only after the extraction procedure were completely clear (data not shown).
The time course for the association and dissociation of [3H]NBI binding to guinea pig brain solubilized extractions is shown in Fig. 3a. The specific binding of 0.45 nM 13H]NBI was 50% complete after approximately 3 min and the binding reaction equilibrium was attained by 20 min. The observed forward rate constant (Aob,) calculated from the slope of the line in Fig. 3b was 0.143 min-' and the dissociation rate constant ( kobtained from the slope of the line in Fig. 3c was 0.046 min-l. K,, as determined using the equation K , = (k,,, -X -I)/ (['HINBI) (Williams et al., 1976), was 2.2 x lo8 min-' M -I . An estimate of the equilibrium dissociation constant (K,) obtained as the ratio k -, / k , was 0.21 nM.
The saturation isotherm and Scatchard analysis of [3H]NBI binding to soluble guinea pig brain ex-  Pharmacology of binding Table 1 shows the pharmacology of [3H]NBI binding in rat and guinea pig brain extracts and that of [3H]CHA binding in guinea pig brain extracts. NBI was a potent displacer of [3H]NBI binding in both guinea pig and rat preparations with K , values in the low nanomolar range. The potent adenosine uptake inhibitors dilazep. hexobendine. and dipyridamole were all powerful inhibitors of [3H]NBI binding in guinea pig preparations with low nanomolar K , values. Of the three uptake inhibitors only dilazep was a potent displacer of binding in both rat and guinea pig extracts. Hexobendine and dipyridamole had a much lower affinity for the soluble rat sites showing K , values of 312.5 and 1,084 nM, respectively. The order of potency of these compounds, however, was similar in both spccies (dilazep, hexobendine, dipyridamole).
Adenosine was a much poorer displacer of [3H1NBI binding in both species with K , values in the high micromolar range. The adenosine receptor agonists CHA and L-PIA displayed K , values of about 1 p M whereas caffeine was a very poor inhibitor in both species with K , values well 'i ' b ove 400 F M . Diazepam, a benzodiazepine, had low micromolar K , values in both species as did the dihydropyridine Ca2 + antagonist nimodipine. Verapamil, a nondihydropyridine Ca'+ antagonist. had a much lower affinity for [3H]NBI sites than nimodipine. In

DISCUSSION
It is becoming quite clear that adenosine serves as a major modulator of physiologic function in the cardiovascular system (Berne, 1980) and in both the peripheral (Burnstock, 1975) and central nervous system (Phillis and Wu. 1981). The ubiquitous presence of this purine, and its role in many metabolic processes has. however, made it difficult to characterize the mechanism of adenosine action in brain.
[3HlNBI binding has been a useful probe for adenosine uptake sites in erythrocytes and HeLa cells because occupation of these sites by L-'H]NBI correlates well with inhibition of nucleoside transport and because this high-affinity binding is competit ivel y displaced by other potent nucleoside transport inhibitors (Cass et al., 1974;Lauzon andPaterson. 1977: Jarvis andYoung, 1980). Similar studies using rat brain tissue (Marangos et al., 1982) and guinea pig cerebral cortical tissue (Hammond and Clanachan, 1982 show ['HINBI to be a good probe for central adenosine uptake sites as well. This study demonstrates that methods used to solubilize brain adenosine receptors (Bruns et al., 1983) can also be used to solubilize adenosine uptake sites from brain using ['HINBI as a probe. These sites, solubilized from guinea pig and rat brains, retain characteristics similar to those reported for [3H]NBI binding sites in insoluble brain membrane preparations from these respective species (Hammond andClanachan, 1982, 1983;Marangos et al., 1982).
Using the procedure described by Bruns et al. we obtained good yields of solubilized adenosine receptors and uptake sites. However, this procedure gave cloudy preparations due to the presence of glycerol in the solubilization buffer, and brought into question the solubility of the preparation. Even though our preparations were centrifuged at 145,000 g for I h, the density of glycerol in our samples affected the centrifugation process. Thus, it was uncertain whether the binding sites were solubilized. Removing glycerol from the solubilizing buffer gave us a clear preparation that still contained active binding sites but lost its activity rapidly. By extracting the sites without any glycerol and then reconstituting the supernatant with glycerol after the extraction, we were able to obtain clear, soluble preparations that were also stable. To ensure solubility, all extractions were passed through a 0.22 p M filter before assaying. The use of glycerol in this manner was necessary for stability and is thus recommended for such extractions.
In the present study, we found that [3H]NBI binding sites solubilized from rat and guinea pig brains had similar kinetic binding parameters. The binding was rapid in both cases and the KD value for [3H]NBI binding to rat brain extracts as determined by Scatchard mass law analysis (KD = 0.12 nM) was similar to K , values for guinea pig soluble sites as determined separately by Scatchard (KD = 0.13 nM) and kinetic analyses (KD = 0.21 nM). The total number of binding sites was similar in both cases (rat, B,,, = 133 fmol/mg protein; guinea pig, B,,, = 150 fmol/mg protein) and linear Scatchard plots indicated the presence of only one class of binding site in both species.
In binding displacement studies NBI was found to be the most potent inhibitor of ['HINBI binding to soluble extracts from both species. In guinea pig preparations the coronary vasodilators dilazep, hexobendine, and dipyridamole were all very potent, competitive inhibitors of ['HINBI binding. These compounds produce their dilatory effect via an inhibition of adenosine uptake leading to the accumulation of endogenous adenosine (Sano, 1974). The K , values reported here for soluble guinea pig sites are similar to those reported for inhibition of [3H]NBI binding in human erythrocytes (Clanachan et al., 1981), HeLa cells (Paterson et al., 1980). and dog heart and brain membrane preparations (Marangos et al., 1984). In rat brain soluble extracts, however, of the three adenosine uptake inhibitors, only dilazep was a strong displacer of ['HINBI binding. Hexobendine and dipyridamole maintained the low affinity for soluble rat ['HINBI binding sites that they display in rat insoluble brain membrane preparations (Marangos et at., 1982: Patel et al.. 1982bWu and Phillis, 1982). Although the reason for their markedly lower potency in rat brain is at present unclear, it is important to note that the order of potency of these uptake inhibitors for displacing ['HINBI binding (dilazep > hexobendine > dipyridamole) was the same in extractions from both species.
It has been suggested that part of the anxiolytic actions of the benzodiazepines may involve inhibition of adenosine uptake (Wu et al., 1981). In our soluble preparations diazepam, which has a very high affinity for benzodiazepine binding sites ( K , value of 7.4 nM) (Mohler and Okada, 1978) showed a much lower affinity for the ['HINBI binding sites extracted from rat and guinea pig brains ( K , values of 6 p M and 9 p M , respectively). These data suggest that if diazepam does indeed inhibit adenosine uptake, it probably does not do so by interacting at the adenosine uptake site that is labeled by NBI.
Conversely, in light of the discrepancies described in rat, compounds inhibiting [3H]NBI binding should be examined for their effects on ["ladenosine uptake as well. An opportunity to examine such a correlation is provided by dihydropyridine calcium antagonist drugs such as nimodipine. Although in our solubilized rat and guinea pig brain extractions nimodipine was only slightly better as an inhibitor of [3H]NBI binding ( K , = 5-6 p M ) than diazepam, this and other dihydropyridines have been shown to have nanomolar K , values for ['HINBI sites in dog heart and brain (Marangos et at., 1984) and in human brain as well (Verma and Marangos, 1985). It will be interesting to examine the effect of these calcium antagonists on the uptake of ['Hladenosine as well as their ability to modulate adenosine action. Verapamil, a nondihydropyridine calcium antagonist was a far poorer inhibitor of ['HINBI binding.
NBI does not interact with the adenosine receptor (Marangos et al., 1982(Marangos et al., , 1983 and the adenosine analogs CHA and L-PIA showed poor affinity for soluble [3H]NBI binding. In addition. when the binding of the adenosine agonist ['HICHA to soluble guinea pig brain extracts was examined, a much different kinetic profile was obtained than that for [3H]NBI binding. ['HICHA is known to label adenosine A, receptor sites, and the biphasic Scatchard plot as well as the two K, values determined for the lowand high-affinity soluble [3H]CHA binding sites in our study are similar to those reported previously for insoluble rat brain preparations (Patel et al., 1982a: Marangos et at., 1983. L-PIA and CHA, in contrast to their poor action at the ['HINBI site, were found to be highly potent inhibitors of ['HICHA binding to soluble extractions. NBI and the other adenosine uptake inhibitors, on the other hand, were very poor displacers of ['HICHA binding. These data indicate that [3H]CHA is a rather specific probe for the solubilized adenosine receptor, just as ['HINBI appears to be a very specific probe for solubilized adenosine uptake sites.
The availability of such distinct probes for adenosine receptors and uptake sites and the ability to use separate and simple assays to study both sites should greatly enhance the understanding of the adenosine system in brain as well as aid in the development of new drugs to affect this system. Solubilization of both sites as described in this study will help in the isolation and further characterization of these two sites.