125I] N6-p-Hydroxyphenylisopropyladenosine, a new ligand for Ri adenosine receptors.

Abstract

N6-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[125I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize Ri adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37 degrees C was of high affinity with an equilibrium dissociation constant KD of 0.48 nmol/l and was saturable with 0.23 pmol of [125I]HPIA per mg of protein. The rate constant of association, k1, was 3.25 x 10(8) l mol-1 min-1 and that of dissociation, k2 0.0110 min-1 yielding at t1/2 of 63 min. In competition experiments the (-)isomer of N6-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for binding sites. Specific binding was most effectively displaced by N6-cyclohexyladenosine (CHA, ki=0.26 nmol/l), (-)PIA (ki= 0.33 nmol/l) and HPIA (ki=0.52 nmol/l), whereas 5'-N-ethylcarboxamidoadenosine (NECA, ki=1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had ki values between 5-43 mumol/l. Binding of [125I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [125I]HPIA labels Ri adenosine receptors in rat brain membranes.