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Tbe 2',3'-dideoxy analogue of the potent A\(_1\) receptor agonist, N\(^6\)-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A\(_1\) adenosine receptor. In sturlies on adenylate cyclase 2',3'-dideoxy-N\(^6\)-cyclohexyladenosine (ddCHA) did not show agonist properties at A\(_1\) or at A\(_2\) receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A\(_1\) receptors with a K\(_i\) value of 13 \(\mu\)M. ddCHA competed for the binding of the selective A1 receptor antagonist, [\(^3\) HJ8-cyclopentyl-1,3-dipropylxantbine ([\(^3\)H]DPCPX), to rat brain membranes with a K\(_i\) value of 4.8 \(\mu\)M; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A\(_1\) receptor for the cx- and the natural ß-anomer of adenosine, the cx-anomer of ddCHA showed a comparable affinity for the A\(_1\) receptor (K\(_i\) value 13.9 \8\mu\)M). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required foragonist activity at and high affinity binding to A\(_1\) adenosine receptors and for the distinction between the cx- and ß-forms.
2-Chloro-N\(^6\)-cyclopentyladenosine: a highly selective agonist at A\(_1\) adenosine receptors
(1988)
2-Chloro-N\(^6\)-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for At adenosine receptors. Binding of [\(^3\)H]PIA to A1 receptors of rat brain membranes was inhibited by CCP A with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N\(^6\)-cyclopentyladenosine (CPA). Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A\(_1\)-selectivity of CCPA. CCP A inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A\(_1\) receptor, with an IC\(_{50}\)-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC\(_{50}\)-value of 3500 nM. The more than 100-fold A\(_1\)-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A\(_1\) adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard At receptor agonist R-N\(^6\) -phenylisopropyladenosine (R-PIA). CCP A represents the agonist with the highest selectivity for A\(_1\) receptors reported so far.
Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]- 1-propanesulfonate) and the solubilized extract subjected to gel ftltration. Binding of the adenosine receptor agonist [\(^3\)H]NECA (5'-N-ethylcarboxamidoadenosine) was measured to the eluted fractions. Two [\(^3\)H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [\(^3\)H]NECA binding activity eluted from the column. It bound [\(^3\)H]NECA in a reversible, saturable and GTPdependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [\(^3\)H]NECA to the frrst peak with a pharmacological proftle characteristic for the A\(_2\) adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [\(^3\)H]NECA binding to the second, major peak. These results suggest that a solubilized A\(_2\) receptor-Gs protein complex of human platelets can be separated from other [\(^3\)H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human plate1ets.
The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A\(_1\) adenosirre receptors were examined and conipared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A\(_1\) adenosirre receptors and the stimulationvia A\(_2\) adenosirre receptors. The K\(_i\)-values of this antagonism were 0.45 nM at the A\(_1\) receptor of rat fat cells, and 330 nM at the A\(_2\) receptor of human platelets, giving a more than 700-fold A\(_1\)-selectivity. A similar A\(_1\)-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMPphosphodiesterase activity of human platelets. [\(^3\)H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A\(_1\) receptors in membranes of bovine brain and heart, and rat brain and fat cells (K\(_D\) -values 50-190 pM). Its nonspecific binding was about 1% of total at K\(_D\) , except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [\(^3\)H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A\(_1\) receptor.