Abstract
This study describes experiments investigating the mechanism of activation
of A1 adenosine receptors. Isolated rat fat cells were used as a
cellular model. The A1 receptors of these cells were covalently labeled
with the agonist photoaffinity label R-2-azido-N6-p-hydroxyphenylisopropyladenosine.
The covalent incorporation of the label into the binding subunit
of the receptor was verified by demonstration of specific labeling
of a peptide with Mr = 35,000 by the radioiodinated label. Such covalent
labeling followed by removal of label not covalently bound led to
a concentration-dependent reduction of cellular cAMP levels. This
persistent effect of covalent labeling occurred with an IC50 value
of 9 nM compared to an IC50 value of 0.9 nM for the direct reduction
of cAMP levels by the label. The affinity of the label was determined
in binding experiments. The Ki value of 19 nM was about 20 times
higher than the corresponding IC50 value of cAMP reduction. Finally,
the comparison between covalent binding and its effects suggests
that covalently labeled receptors were fully activated. The data
are interpreted as evidence for a receptor activation according to
the occupancy theory. The analysis of the various concentration-response
curves reveals the presence of spare receptors, which can be demonstrated
by the method of agonist photoaffinity labeling.
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