GONZALEZCALERO, G; CUBERO, A; KLOTZ, KN

UNIV CASTILLA LA MANCHA,FAC CIENCIAS QUIM,DEPT QUIM,E-13071 CIUDAD REAL,SPAIN; UNIV HEIDELBERG,INST PHARMAKOL,W-6900 HEIDELBERG,GERMANY

A(l) adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[H-3]dipropyl-xanthine ([H-3]DPCPX) gave a K(d) value of 0.7 nM and a B(max) value of 82 +/- 13 fmol/mg protein. For the highly A(l)-selective agonist 2-chloro-N6-VH]cyclopentyladenosine ([H-3]CCPA) a K(d) value of 1.7 nM and a B(max) value of 72 +/- 29 fmol/mg protein was estimated. Competition of agonists for [H-3]DPCPX binding gave a pharmacological profile with R-N-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N-I-125-p-hydroxyphenylisopropyladenosine ([I-125]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A(l) receptors labelled in brain membranes. Therefore, coated vesicles contain A(l) adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A(l) receptors in these vesicles are not a processed receptor form. These results confirm that A1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A(l) receptor systems in coated vesicles and in plasma membranes are identical.