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High expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue
ejp ELSEVIER
European Journal of Pharmacology257 (1994) 311-314
Short communication
High expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue Stacey A. Jones-Humble *, Philip F. Morgan Wellcome Research Laboratories, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, NC 27709, USA
(Received 22 March 1994; accepted 25 March 1994)
Abstract
The presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue is reported. Displacement studies using 15 nM [3H]dipyridamole revealed 50-60% of the sites were sensitive to nitrobenzylthioinosine. Non-linear analysis of binding isotherms to estimate the density of nitrobenzylthioinosine-sensitive and -insensitive sites revealed a maximum number of nitrobenzylthioinosine-sensitive binding sites (Bmax) of 395 + 19 fmol/mg protein and a nitrobenzylthioinosine-insensitive Bmax of 3910 + 700 fmol/mg protein (corresponding K d values of 0.1 nM and 114 nM respectively). Thus there are approximately 10 times as many nitrobenzylthioinosine-insensitive sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes. Key words: Adenosine; Nucleoside transporter; Dipyridamole; Nitrobenzylthioinosine; Brain
1. Introduction
Adenosine, an endogenous purine nucleoside, is an important neuromodulator in the central nervous system which acts upon ectocellular adenosine receptors to modulate a large number of neuronal functions (Stone, 1981; Phillis and Wu, 1981). The major route of inactivation of extracellular adenosine is via uptake into cells by nucleoside transporters which translocate adenosine and other nucleosides by an equilibrative facilitated diffusion process (Paterson et al., 1983; Zhang et al., 1993). Considerable evidence suggests that there is a heterogeneity of nucleoside transporters (Deckert et al., 1988). In particular, two predominant sub-sets of nucleoside transporters can be differentiated by their sensitivity to the purine nitrobenzylthioinosine. Thus nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive components to nucleoside transport have been described (Davies and Hambley, 1986).
* Corresponding author. Department of Pharmacology, Wellcome Research Laboratories, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, NC 27709, USA. Tel. (919) 315-3850, fax (919) 315-8890. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00191-9
Nucleoside transporters can be identified using the transport inhibitors nitrobenzylthioinosine and dipyridamole. [3H]Nitrobenzylthioinosine exhibits affinity for only the nitrobenzylthioinosine-sensitive sub-set of nucleoside transporters (Marangos et al., 1982) whereas [3H]dipyridamole exhibits high affinity for both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive transporters (Davies and Hambley, 1986; Marangos and Deckert, 1987). Selective inhibitors of nitrobenzylthioinosine-insensitive nucleoside transporters have not been described to date although the expression of nitrobenzylthioinosine-insensitive sites can be inferred from the difference between [3H]nitrobenzylthioinosine and [3H]dipyridamole labeled sites using saturation isotherms. Studies reveal that there is a large variation in the relative expression of nitrobenzylthioinosine-sensitive and -insensitive dipyridamole binding sites in different organs and tissues (Deckert et al., 1988). There is also considerable interspecies variation, particularly in the expression of the nitrobenzylthioinosine-insensitive dipyridamole binding sites (Plagemann et al., 1988). For example, expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site is high in guinea pig brain (particularly in ependymal tissue) whereas expression of this site in rat brain is not detectable (Deckert et al.,
312
S..4. Jones-Humble, P.F. Morgan / European Journal ()["Pharmacology 257 (1994) 311-314
1988). Initial studies have detected the presence of nitrobenzylthioinosine-sensitive, but not nitrobenzylthioinosine-insensitive, dipyridamole binding sites in postmortem human brain using parietal cortex crude membranes (Deckert et al., 1992). In view of the potential therapeutic importance of determining the presence of nitrobenzylthioinosine-insensitive nucleoside transporters in human tissues we have examined human ependymal membranes for expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites.
liquid scintillation cocktail (Beckman, Fullerton, CA) and counted on a Packard 2000CA Tri-Carb Liquid Scintillation Analyzer (Packard, Downers Grove, IL). 2.3. Displacement binding assays
2. Materials and methods
Filtration binding assays were performed essentially as described above. A volume corresponding to 5 mg of tissue (wet weight) was incubated for 30 min at room temperature with 15 nM [3H]dipyridamole in the presence of 10 /zM nitrobenzylthioinosine. Nonspecific binding (approximately 50% of total binding) was determined in the presence of 10/xM dipyridamole.
2.1. Tissue preparation
2.4. Protein assay
Human ependymal tissue was obtained from eight postmortem brains which were frozen within 4 h of death. The tissue donors were between the ages of 21 and 86 years, four were female (one African-American, three Caucasian) and four were male (all Caucasian). The cause of death was either cardiac or respiratory arrest in each case. Tissue preparation involved diluting the ependymal tissue in 25 volumes of ice-cold 50 mM Tris-HCl buffer, pH 7.3 then homogenizing using a Polytron (setting 7.4) for 15 s. The homogenate was centrifuged at 30000 × g at 4°C for 20 min. The pellet was resuspended, homogenized and centrifuged as before. The pellet was immediately frozen and stored at - 8 0 ° C until used. 2. 2. Saturation binding assays
The pellets were thawed and resuspended in 50 mM Tris-HCl buffer, pH 7.4. A Polytron (setting 7.4) was used to homogenize the tissue before use in filtration binding assays. For nitrobenzylthioinosine binding a volume corresponding to 10 mg of tissue (wet weight) was incubated for 30 min at room temperature with 0.1-5 nM [3H]nitrobenzylthioinosine (27.3 Ci/mmol, N E N / D u P o n t , Boston, MA). Nonspecific binding was determined in the presence of 5 /xM nitrobenzylthioinosine (Research Biochemicals, Natick, MA) and represented 10% of total binding at 0.1 nM free [SH]nitrobenzylthioinosine. For dipyridamole binding a volume corresponding to 5 mg of tissue (wet weight) was incubated for 30 rain at room temperature with 0.1-880 nM [3H]dipyridamole (58 Ci/mmol, Moravek Biochemicals, Brea, CA) in the presence of 10 p,M nitrobenzylthioinosine. Nonspecific binding was determined in the presence of 10 /xM dipyridamole (Research Biochemicals) and represented 63% of total binding at 16.6 nM free [3H]dipyridamole. Samples were filtered through G F / B filters soaked in 0.1% bovine serum albumin using a Brandel Cell Harvester. Samples were washed 3 times with 3 ml ice-cold 50 mM Tris pH 7.4. Filters were placed in 10 ml ReadySafe
The amount of protein present in the binding assays was determined by protein analysis using the Bio-Rad Protein Assay system (Bio-Rad, Richmond, CA). 2.5. Data analysis
Equilibrium dissociation rates (K d) and maximum number of binding sites (Bmax) were calculated using Lundon-1 software (Lundon Software, Chagrin Falls, OH). Curve fit by nonlinear regression using Inplot (GraphPad Software, San Diego, CA).
3. Results
The expression of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyri-
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10-7 10-6 10-5 Nitrobenzylthioinosine (M) Fig. 1. Inhibition of [3H]dipyridamolebinding in human ependymal tissue by nitrobenzylthioinosine.Tissue was incubated with 15 nM [3Hldipyridamoleand the indicated concentration of nitrobenzylthioinosine for 30 min. Results represent mean + S.E.M. of three experiments, each run in duplicate. Curve fit by nonlinear regression using Inplot (GraphPad Software, San Diego, CA) correlation coefficient = 0.990. 1
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SM. Jones-Humble, P.F. Morgan/European Journal of Pharmacology 257 (1994) 311-314 Table 1 K d and Bmax values from [3H]nitrobenzylthioinosine and [3H]dipyridamole saturation binding studies in human brain ependymal tissue Radioligand
Kd
Bmax
[3H]Nitrobenzylthioinosine a
110 + 26 pM
[3H]Dipyridamole b
114+ 7 nM
395 + 19 fmol/mg protein 3.91+ 0.7 pmol/mg protein
Analysis by Lundon-1 software. a Values represent the average and S.E.M. of three separate 6-12 point saturation experiments each run in duplicate. b [3H]Dipyridamole binding done in the presence of 10 mM nitrobenzylthioinosine. Values represent the average and S.E.M. of four separate 6-12 point saturation experiments each run in duplicate.
damole binding sites in postmortem human ependymal tissue was identified. Using nitrobenzylthioinosine to displace [3H]dipyridamole from nitrobenzylthioinosinesensitive sites revealed that only 50-60% of the sites specifically labeled by 15 nM [3H]dipyridamole were displaced by nitrobenzylthioinosine in concentrations up to 10/xM (i.e., nitrobenzylthioinosine-sensitive) (Fig. 1). Hence 40-50% of specific [3H]dipyridamole binding is refractory to inhibition by nitrobenzylthioinosine (i.e., nitrobenzylthioinosine-insensitive). The relative density of nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive binding sites in ependymal membranes was estimated from saturation isotherms. Non-linear analysis of binding isotherms using [3H]nitrobenzylthioinosine to estimate the density of nitrobenzylthioinosine-sensitive sites revealed a maximum number of binding sites (Bmax) of 395 _+ 19 fmol/mg protein (Table 1). Non-linear analysis of binding isotherms using [3H]dipyridamole (in the presence of 10 mM nitrobenzylthioinosine to mask nitrobenzylthioinosine-sensitive sites) to estimate the density of nitrobenzylthioinosine-insensitive sites revealed a Bmax of 3910 _+ 700 fmol/mg protein (Table 1). Therefore there are approximately 10 times as many nitrobenzylthioinosine-insensitive [3H]dipyridamole sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes.
4. Discussion
The present results reveal the presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue. Nitrobenzylthioinosine-sensitive dipyridamole binding sites appear to be ubiquitous. They have previously been detected in postmortem human parietal cortex tissue (Deckert et al., 1992) and in human peripheral tissues such as erythrocytes (Woffendin and Plagemann, 1987). In con-
313
trast, nitrobenzylthioinosine-insensitive dipyridamole binding sites were not detected in postmortem human parietal cortex tissue (Deckert et al., 1992), nor have they been detected in peripheral human tissues such as erythrocytes (Woffendin and Plagemann, 1987). The findings of previous studies (Hammond and Clanachan, 1985; Morgan and Marangos, 1987) point to the considerable species variation in the expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site. Several studies have not detected this site in rat brain (Hammond and Clanachan, 1985; Verma and Marangos, 1985; Marangos and Deckert, 1987; Deckert et al., 1988), whereas expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site is reported to be high in guinea pig brain (especially in the ependyma) (Deckert et al., 1988). Therefore the guinea pig may be a better model with which to study the nitrobenzylthioinosine-insensitive dipyridamole binding site. The present study reveals a high density of nitrobenzylthioinosine-insensitive [3H]dipyridamole binding sites in human ependymal tissue (ratio of approximately 10:1 compared to nitrobenzylthioinosine-sensitive binding sites). The nitrobenzylthioinosine-insensitive [3H]dipyridamole binding sites may represent a class of nucleoside transporters which are distinct from the ubiquitous nitrobenzylthioinosine-sensitive transporters although these sites could also represent a separate binding site for dipyridamole that is unrelated to inhibition of nucleoside transport. In tissues largely comprised of nitrobenzylthioinosine-sensitive dipyridamole binding sites (e.g., erythrocytes and rat brain) both dipyridamole and nitrobenzylthioinosine (10 -5 M) can inhibit most of the specific uptake of adenosine (Morgan and Marangos, 1987). In tissue comprised of nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive binding sites (e.g., guinea pig brain) nitrobenzylthioinosine (10 -5 M) only partially inhibits specific adenosine uptake while dipyridamole inhibits most of the specific uptake (Morgan and Marangos, 1987). It has been suggested that nitrobenzylthioinosine-sensitive and -insensitive binding sites may subserve different components of nucleoside uptake (Morgan and Marangos, 1987; Deckert et al., 1988). If this is the case then human ependymal tissue, which expresses both types of dipyridamole binding sites, may express multiple nucleoside transport proteins which are differentially sensitive to nitrobenzylthioinosine and, to a lesser extent, dipyridamole. If these sites represent two distinct classes or subtypes of nucleoside transporter they may be useful therapeutic targets for modulation of ependymal cell function (e.g., modulation of edema). This is a particularly attractive possibility since many peripheral tissues appear to lack nitrobenzylthioinosine-insensitive dipyridamole binding sites and therefore specific ligands for the nitroben-
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zylthioinosine-insensitive nucloside transporter may be devoid of many peripheral (e.g., local cardiovascular) actions.
Acknowledgements The authors thank Dr. Michael Durcan and Dr. Richard F. Cox for helpful discussions during preparation of the manuscript.
References Davies, L.P. and J.W. Hambley, 1986, Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: evidence for nitrobenzylthioinosine-sensitive and insensitive sites'?., Neurochem. Int. 8, 103. Deckert, J., P.F. Morgan and P.J. Marangos, 1988, Adenosine uptake site heterogeneity in the mammalian CNS? uptake inhibitors as probes and potential neuropharmaceuticals, Life Sci. 42, 1331. Deckert, J., A. Hennemann, B. Bereznai, W. Gsell, M. Gotz, H. Fritze, H. Beckmann and P. Riederer, 1992, Heterogeneity of adenosine transporters as basis for CNS-specific adenosine transport inhibitors?, Soc. Neurosci. Abstr. 18, 997. Hammond, J.R. and A.S. Clanachan, 1985, Species differences in the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system in mammalian central nervous system membranes: evidence for interconvertible conformations of the binding site/transporter complex, J. Neurochem. 45, 527.
Marangos, P.J., J. Patel, R. Clark-Rosenberg and A.M, Martino, 1982, [3H]Nitrobenzylthioinosine binding as a probe for the study of adenosine uptake sites in brain, J. Neurochem. 39, 184. Marangos, P.J. and .1. Deckert, 1987, [3H]Dipyridamole binding to guinea pig brain membranes: possible heterogeneity of central adenosine uptake sites, J. Neurochem. 48, 1231. Morgan, P.F. and P.J. Marangos, 1987, Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptosomes, Neurochem. Int. 11,339. Paterson, A.R.P., E.S. Jakobs, E.R. Hawley, N.W. Fu, M.J. Robins and C.E. Cass, 1983, Inhibition of nucleoside transport, in: Regulatory Function of Adenosine, eds. R.M. Berne, T.W. Rail and R. Rubio (Martinus Nijhoff, The Hague) p. 203. Philis, J.W. and P.H. Wu, 1981, The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol. 16, 187. Plagemann, P.G.W., R.M. Wohlhueter and C. Woffendin, 1988, Nucleoside and nucleobase transport in animal cells, Biochim. Biophys. Acta 947, 405. Stone, T.W., 1981, Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience 6, 523. Verma, A. and P.J. Marangos, 1985, Nitrobenzylthioinosine binding in brain: an interspecies study, Life Sci. 36, 283. Woffendin, C. and P.G. Plagemann, 1987, Interaction of [3H]dipyridamole with the nucleoside transporters of human erythrocytes and cultured animal cells, J. Membr. Biol. 98, 89. Zhang, G., P.H. Franklin and T.H. Murray, 1993, Manipulation of endogenous adenosine in the rat prepiriform cortex modulates seizure susceptibility, J. Pharmacol. Exp. Ther. 264, 1415.
European Journal of Pharmacology257 (1994) 311-314
Short communication
High expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue Stacey A. Jones-Humble *, Philip F. Morgan Wellcome Research Laboratories, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, NC 27709, USA
(Received 22 March 1994; accepted 25 March 1994)
Abstract
The presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue is reported. Displacement studies using 15 nM [3H]dipyridamole revealed 50-60% of the sites were sensitive to nitrobenzylthioinosine. Non-linear analysis of binding isotherms to estimate the density of nitrobenzylthioinosine-sensitive and -insensitive sites revealed a maximum number of nitrobenzylthioinosine-sensitive binding sites (Bmax) of 395 + 19 fmol/mg protein and a nitrobenzylthioinosine-insensitive Bmax of 3910 + 700 fmol/mg protein (corresponding K d values of 0.1 nM and 114 nM respectively). Thus there are approximately 10 times as many nitrobenzylthioinosine-insensitive sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes. Key words: Adenosine; Nucleoside transporter; Dipyridamole; Nitrobenzylthioinosine; Brain
1. Introduction
Adenosine, an endogenous purine nucleoside, is an important neuromodulator in the central nervous system which acts upon ectocellular adenosine receptors to modulate a large number of neuronal functions (Stone, 1981; Phillis and Wu, 1981). The major route of inactivation of extracellular adenosine is via uptake into cells by nucleoside transporters which translocate adenosine and other nucleosides by an equilibrative facilitated diffusion process (Paterson et al., 1983; Zhang et al., 1993). Considerable evidence suggests that there is a heterogeneity of nucleoside transporters (Deckert et al., 1988). In particular, two predominant sub-sets of nucleoside transporters can be differentiated by their sensitivity to the purine nitrobenzylthioinosine. Thus nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive components to nucleoside transport have been described (Davies and Hambley, 1986).
* Corresponding author. Department of Pharmacology, Wellcome Research Laboratories, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, NC 27709, USA. Tel. (919) 315-3850, fax (919) 315-8890. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00191-9
Nucleoside transporters can be identified using the transport inhibitors nitrobenzylthioinosine and dipyridamole. [3H]Nitrobenzylthioinosine exhibits affinity for only the nitrobenzylthioinosine-sensitive sub-set of nucleoside transporters (Marangos et al., 1982) whereas [3H]dipyridamole exhibits high affinity for both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive transporters (Davies and Hambley, 1986; Marangos and Deckert, 1987). Selective inhibitors of nitrobenzylthioinosine-insensitive nucleoside transporters have not been described to date although the expression of nitrobenzylthioinosine-insensitive sites can be inferred from the difference between [3H]nitrobenzylthioinosine and [3H]dipyridamole labeled sites using saturation isotherms. Studies reveal that there is a large variation in the relative expression of nitrobenzylthioinosine-sensitive and -insensitive dipyridamole binding sites in different organs and tissues (Deckert et al., 1988). There is also considerable interspecies variation, particularly in the expression of the nitrobenzylthioinosine-insensitive dipyridamole binding sites (Plagemann et al., 1988). For example, expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site is high in guinea pig brain (particularly in ependymal tissue) whereas expression of this site in rat brain is not detectable (Deckert et al.,
312
S..4. Jones-Humble, P.F. Morgan / European Journal ()["Pharmacology 257 (1994) 311-314
1988). Initial studies have detected the presence of nitrobenzylthioinosine-sensitive, but not nitrobenzylthioinosine-insensitive, dipyridamole binding sites in postmortem human brain using parietal cortex crude membranes (Deckert et al., 1992). In view of the potential therapeutic importance of determining the presence of nitrobenzylthioinosine-insensitive nucleoside transporters in human tissues we have examined human ependymal membranes for expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites.
liquid scintillation cocktail (Beckman, Fullerton, CA) and counted on a Packard 2000CA Tri-Carb Liquid Scintillation Analyzer (Packard, Downers Grove, IL). 2.3. Displacement binding assays
2. Materials and methods
Filtration binding assays were performed essentially as described above. A volume corresponding to 5 mg of tissue (wet weight) was incubated for 30 min at room temperature with 15 nM [3H]dipyridamole in the presence of 10 /zM nitrobenzylthioinosine. Nonspecific binding (approximately 50% of total binding) was determined in the presence of 10/xM dipyridamole.
2.1. Tissue preparation
2.4. Protein assay
Human ependymal tissue was obtained from eight postmortem brains which were frozen within 4 h of death. The tissue donors were between the ages of 21 and 86 years, four were female (one African-American, three Caucasian) and four were male (all Caucasian). The cause of death was either cardiac or respiratory arrest in each case. Tissue preparation involved diluting the ependymal tissue in 25 volumes of ice-cold 50 mM Tris-HCl buffer, pH 7.3 then homogenizing using a Polytron (setting 7.4) for 15 s. The homogenate was centrifuged at 30000 × g at 4°C for 20 min. The pellet was resuspended, homogenized and centrifuged as before. The pellet was immediately frozen and stored at - 8 0 ° C until used. 2. 2. Saturation binding assays
The pellets were thawed and resuspended in 50 mM Tris-HCl buffer, pH 7.4. A Polytron (setting 7.4) was used to homogenize the tissue before use in filtration binding assays. For nitrobenzylthioinosine binding a volume corresponding to 10 mg of tissue (wet weight) was incubated for 30 min at room temperature with 0.1-5 nM [3H]nitrobenzylthioinosine (27.3 Ci/mmol, N E N / D u P o n t , Boston, MA). Nonspecific binding was determined in the presence of 5 /xM nitrobenzylthioinosine (Research Biochemicals, Natick, MA) and represented 10% of total binding at 0.1 nM free [SH]nitrobenzylthioinosine. For dipyridamole binding a volume corresponding to 5 mg of tissue (wet weight) was incubated for 30 rain at room temperature with 0.1-880 nM [3H]dipyridamole (58 Ci/mmol, Moravek Biochemicals, Brea, CA) in the presence of 10 p,M nitrobenzylthioinosine. Nonspecific binding was determined in the presence of 10 /xM dipyridamole (Research Biochemicals) and represented 63% of total binding at 16.6 nM free [3H]dipyridamole. Samples were filtered through G F / B filters soaked in 0.1% bovine serum albumin using a Brandel Cell Harvester. Samples were washed 3 times with 3 ml ice-cold 50 mM Tris pH 7.4. Filters were placed in 10 ml ReadySafe
The amount of protein present in the binding assays was determined by protein analysis using the Bio-Rad Protein Assay system (Bio-Rad, Richmond, CA). 2.5. Data analysis
Equilibrium dissociation rates (K d) and maximum number of binding sites (Bmax) were calculated using Lundon-1 software (Lundon Software, Chagrin Falls, OH). Curve fit by nonlinear regression using Inplot (GraphPad Software, San Diego, CA).
3. Results
The expression of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyri-
120
c-
100
15 _c m .o
80
E
60
L+-
~D
50
13 U_
~ I c¢3
40 20 g_
........
i
........
i
, .......
i
i ,~,,.,I
~ ll,..I
10-7 10-6 10-5 Nitrobenzylthioinosine (M) Fig. 1. Inhibition of [3H]dipyridamolebinding in human ependymal tissue by nitrobenzylthioinosine.Tissue was incubated with 15 nM [3Hldipyridamoleand the indicated concentration of nitrobenzylthioinosine for 30 min. Results represent mean + S.E.M. of three experiments, each run in duplicate. Curve fit by nonlinear regression using Inplot (GraphPad Software, San Diego, CA) correlation coefficient = 0.990. 1
lo
10-g
lO-S
SM. Jones-Humble, P.F. Morgan/European Journal of Pharmacology 257 (1994) 311-314 Table 1 K d and Bmax values from [3H]nitrobenzylthioinosine and [3H]dipyridamole saturation binding studies in human brain ependymal tissue Radioligand
Kd
Bmax
[3H]Nitrobenzylthioinosine a
110 + 26 pM
[3H]Dipyridamole b
114+ 7 nM
395 + 19 fmol/mg protein 3.91+ 0.7 pmol/mg protein
Analysis by Lundon-1 software. a Values represent the average and S.E.M. of three separate 6-12 point saturation experiments each run in duplicate. b [3H]Dipyridamole binding done in the presence of 10 mM nitrobenzylthioinosine. Values represent the average and S.E.M. of four separate 6-12 point saturation experiments each run in duplicate.
damole binding sites in postmortem human ependymal tissue was identified. Using nitrobenzylthioinosine to displace [3H]dipyridamole from nitrobenzylthioinosinesensitive sites revealed that only 50-60% of the sites specifically labeled by 15 nM [3H]dipyridamole were displaced by nitrobenzylthioinosine in concentrations up to 10/xM (i.e., nitrobenzylthioinosine-sensitive) (Fig. 1). Hence 40-50% of specific [3H]dipyridamole binding is refractory to inhibition by nitrobenzylthioinosine (i.e., nitrobenzylthioinosine-insensitive). The relative density of nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive binding sites in ependymal membranes was estimated from saturation isotherms. Non-linear analysis of binding isotherms using [3H]nitrobenzylthioinosine to estimate the density of nitrobenzylthioinosine-sensitive sites revealed a maximum number of binding sites (Bmax) of 395 _+ 19 fmol/mg protein (Table 1). Non-linear analysis of binding isotherms using [3H]dipyridamole (in the presence of 10 mM nitrobenzylthioinosine to mask nitrobenzylthioinosine-sensitive sites) to estimate the density of nitrobenzylthioinosine-insensitive sites revealed a Bmax of 3910 _+ 700 fmol/mg protein (Table 1). Therefore there are approximately 10 times as many nitrobenzylthioinosine-insensitive [3H]dipyridamole sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes.
4. Discussion
The present results reveal the presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue. Nitrobenzylthioinosine-sensitive dipyridamole binding sites appear to be ubiquitous. They have previously been detected in postmortem human parietal cortex tissue (Deckert et al., 1992) and in human peripheral tissues such as erythrocytes (Woffendin and Plagemann, 1987). In con-
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trast, nitrobenzylthioinosine-insensitive dipyridamole binding sites were not detected in postmortem human parietal cortex tissue (Deckert et al., 1992), nor have they been detected in peripheral human tissues such as erythrocytes (Woffendin and Plagemann, 1987). The findings of previous studies (Hammond and Clanachan, 1985; Morgan and Marangos, 1987) point to the considerable species variation in the expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site. Several studies have not detected this site in rat brain (Hammond and Clanachan, 1985; Verma and Marangos, 1985; Marangos and Deckert, 1987; Deckert et al., 1988), whereas expression of the nitrobenzylthioinosine-insensitive dipyridamole binding site is reported to be high in guinea pig brain (especially in the ependyma) (Deckert et al., 1988). Therefore the guinea pig may be a better model with which to study the nitrobenzylthioinosine-insensitive dipyridamole binding site. The present study reveals a high density of nitrobenzylthioinosine-insensitive [3H]dipyridamole binding sites in human ependymal tissue (ratio of approximately 10:1 compared to nitrobenzylthioinosine-sensitive binding sites). The nitrobenzylthioinosine-insensitive [3H]dipyridamole binding sites may represent a class of nucleoside transporters which are distinct from the ubiquitous nitrobenzylthioinosine-sensitive transporters although these sites could also represent a separate binding site for dipyridamole that is unrelated to inhibition of nucleoside transport. In tissues largely comprised of nitrobenzylthioinosine-sensitive dipyridamole binding sites (e.g., erythrocytes and rat brain) both dipyridamole and nitrobenzylthioinosine (10 -5 M) can inhibit most of the specific uptake of adenosine (Morgan and Marangos, 1987). In tissue comprised of nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive binding sites (e.g., guinea pig brain) nitrobenzylthioinosine (10 -5 M) only partially inhibits specific adenosine uptake while dipyridamole inhibits most of the specific uptake (Morgan and Marangos, 1987). It has been suggested that nitrobenzylthioinosine-sensitive and -insensitive binding sites may subserve different components of nucleoside uptake (Morgan and Marangos, 1987; Deckert et al., 1988). If this is the case then human ependymal tissue, which expresses both types of dipyridamole binding sites, may express multiple nucleoside transport proteins which are differentially sensitive to nitrobenzylthioinosine and, to a lesser extent, dipyridamole. If these sites represent two distinct classes or subtypes of nucleoside transporter they may be useful therapeutic targets for modulation of ependymal cell function (e.g., modulation of edema). This is a particularly attractive possibility since many peripheral tissues appear to lack nitrobenzylthioinosine-insensitive dipyridamole binding sites and therefore specific ligands for the nitroben-
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zylthioinosine-insensitive nucloside transporter may be devoid of many peripheral (e.g., local cardiovascular) actions.
Acknowledgements The authors thank Dr. Michael Durcan and Dr. Richard F. Cox for helpful discussions during preparation of the manuscript.
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