Comparison of A1_1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling

Radioligand binding to A$_1$ adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1 ,3-[$^3$H]dipropylxanthine ([$^3$H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [$^3$H]DPCPX binding showed that the most potent compounds were DPCPX with K$_i$ values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K$_i$ values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radio Iigand 2-chloro-N$^6$ -[$^3$H]cyclopen tyladenosine ([$^3$H]CCP A) were less pronounced, rauging from a K$_D$ value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [$^3$H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N$^6$-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K$_D$ values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A$_1$ receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A$_1$ receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences

Characterization of the K+^+-channel-coupled adenosine receptor in guinea pig atria

In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on 86Rb + -efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1 ,3-[$^3$H]dipropylxanthine ([$^3$H]DPCPX) to atrial membrane preparations. The rate of \8^{86}\)Rb$^+$ -effiux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC50-values for 2-chloro-N$^6$-cyclopentyladenosine (CCPA), R-N$^6$-phenylisopropyladenosine (R-PIA), 5'-Nethylcarboxamidoadenosine (NECA), and S-N$^6$-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 $\mu$M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K$_B$-value of 8.1 nM, indicating competitive antagonism. [$^3$H]DPCPX showed a saturable binding to atrial membranes with a Bmax·value of 227 fmol/mg protein and a K$_D$-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K$^+$ channel-coupled adenosine receptor in guinea pig atria is of an A$_1$ subtype