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Characteristics of High Affinity and Low Affinity Adenosine Binding Sites in Human Cerebral Cortex

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Purine and Pyrimidine Metabolism in Man V

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 195B))

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Summary

The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. The ligands used were 2-chloro[8-3H]adenosine and N6-[adenine-2,8-3 H]cyclohexyladenosine.

Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The Kd calculated for chloroadenosine by Scatchard analysis of equilibrium data was 280 nM, with a Bmax of 1.6 pmoles/mg protein, suggesting a single class of binding sites. The specificity of chloroadenosine binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach, the apparent Kd was estimated to be O.74 μ-M for 5′-N-ethylcarboxamideadenosine, 1 ′M cyclohexyladenosine, and 13 ′M for N6-(L-2-phenylisopropyl)adenosine. Isobutylmethylxanthine and theophylline, receptor antagonists, had apparent Kd values of 84 ′M and 105 ′M, respectively. Hill slope factors ranged from O.3 to O.6. Chloroadenosine binding to human brain cortical membranes approached equilibrium at 90 minutes, with a T 1/2, of 10 minutes. The kob was O.080 min−1 and the k1 was 7.5 x 104 min−1 M−1. Reversibility of chloroadenosine binding at equilibrium was completed at approximately 10 minutes with a k2 value of 0.074 min −1. The Kd calculated from the rate constants was 990 nM.

Cyclohexyladenosine binding was concentration dependent. The Kd calculated for cyclohexyladenosine via Scatchard analysis of equilibrium data was 5 nM with a Bmax of O.35 pmoles/mg protein. Cyclohexyladenosine binding was displaced by 3 known receptor agonists: N6 -(L-2-phenyliso propyl)adenosine (Kd 4 nM), 2-chloroadenosine (Kd 10 nM) and 5′-N-ethyl-carboxamideadenosine (Kd 6 nM). The apparent Kd values for the agonists were 1 to 3 orders of magnitude lower with this ligand as compared to radioactive chloroadenosine. Binding was also displaced by 2 known antagonists, isobutylmethylxanthine and theophylline, with apparent Kd values of 4 μM and 8 μM, respectively. Hill slope factors ranged from 0.5 to 0.8.

Our data support the existence of two adenosine binding sites in human cortex compatable with the low affinity (A2) and high affinity (A1) adenosine receptors.

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Authors and Affiliations

  1. From the Human Purine Research Center Department of Internal Medicine and Biological Chemistry, Clinical Research Center, University Hospital, The University of Michigan, Ann Arbor, Michigan, 48109, USA

    David John & Irving H. Fox

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  1. David John
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  2. Irving H. Fox
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Editors and Affiliations

  1. University of California, San Diego La Jolla, California, USA

    W. L. Nyhan

  2. Scripps Clinic and Research Foundation, La Jolla, California, USA

    L. F. Thompson

  3. M. R. C. Clinical Research Center, Harrow, UK

    R. W. E. Watts

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© 1986 Plenum Press, New York

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John, D., Fox, I.H. (1986). Characteristics of High Affinity and Low Affinity Adenosine Binding Sites in Human Cerebral Cortex. In: Nyhan, W.L., Thompson, L.F., Watts, R.W.E. (eds) Purine and Pyrimidine Metabolism in Man V. Advances in Experimental Medicine and Biology, vol 195B. Springer, New York, NY. https://doi.org/10.1007/978-1-4684-1248-2_10

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  • DOI: https://doi.org/10.1007/978-1-4684-1248-2_10

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