Summary
The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCPA was prepared with a specific radioactivity of 1.58 TBq/mmol (43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinityK D-value of 0.2 nmol/l. In the presence of GTP aK D-value of 13 nmol/l was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confirmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgCl2, as has been shown for the agonist [3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A KD-value of 0.4 nmol/l and aB max-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmol/l, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [3H]CCPA proved to be a useful agonist radioligand for characterization of A1 adenosine receptors also in tissues with very low receptor density.
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Abbreviations
- CHA:
-
N6-cyclopenyadenosine
- CPA:
-
N6-cy-clopentyladen,osine
- CCPA:
-
2-chloro-N6-cyclopentyladenosine
- CCCPA:
-
2-chloro-5′-chloro-5′-deoxy-N6-cyclopentyladenosine;
- CHAPS:
-
3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate
- DPCPX:
-
8-cyclopentyl-1,3-dipropylxanthine
- NECA:
-
N-ethylcarboxamidoadenosine
- PEI:
-
polyethylenimine
- PIA:
-
N6-phenylisopropyladenosine
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Klotz, KN., Lohse, M.J., Schwabe, U. et al. 2-Chloro-N6-[3H]cyclopentyladenosine ([3HCCPA) —a high affinity agonist radioligand for A1 adenosine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol. 340, 679–683 (1989). https://doi.org/10.1007/BF00717744
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DOI: https://doi.org/10.1007/BF00717744