Abstract
The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA),
an adenosine derivative with subnanomolar affinity and a 10,000-fold
selectivity for A1 adenosine receptors, has been examined as a new
agonist radioligand. 3HCCPA 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 affinity KD-value
of 0.2 nmol/l. In the presence of GTP a KD-value of 13 nmol/l was
determined for the low affinity state for agonist binding. Competition
of several adenosine receptor agonists and antagonists for 3HCCPA
binding to rat brain membranes confirmed binding to an A1 receptor.
Solubilized A1 receptors bound 3HCCPA 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 3HN6-phenylisopropyladenosine (3HPIA). 3HCCPA 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 a Bmax-value of 16 fmol/mg protein was determined in these membranes.
In human platelet membranes no specific binding of 3HCCPA was measured
at concentrations up to 400 nmol/l, indicating that A2 receptors
did not bind 3HCCPA. Based on the subnanomolar affinity and the
high selectivity for A1 receptors 3HCCPA 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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