Labelling of Ri adenosine receptors in rat fat cell membranes with (-)-[125iodo]N6-hydroxyphenylisopropyladenosine.

A new adenosine analogue, (-)-iodo-N6-phydroxyphenylisopropyladenosine [(-)-IHPIA], has been developed for radioligand binding studies of Ri adenosine receptors. In addition, the effects of (-)IHPIA on adenosine-mediated responses of rat fat cells have been characterized. (-)IHPIA is slightly less potent at Ri adenosine receptors than (-)N6-phenylisopropyladenosine [(-)PIA] as assessed by adenylate cyclase and lipolysis studies. (-)IHPIA inhibited basal adenylate cyclase activity with an IC50 of 60 nmol/l compared to an IC50 of 16.3 nmol/l for (-)PIA. (-)PIA and (-)IHPIA inhibited adenosine deaminase-stimulated lipolysis of intact rat fat cells with an IC50 of 0.55 and 3.6 nmol/l. The potency of (-)N6-phydroxyphenylisopropyladenosine [(-)HPIA] was intermediate. (-)HPIA has been labelled with carrier-free Na[125I] to very high specific activity (2,175 Ci/mmol) and used as agonist radioligand in binding studies of Ri adenosine receptors. The binding of (-)[125I]HPIA was saturable, reversible and stereospecific. Saturation analysis revealed two affinity states with dissociation constants (KD) of 0.7 and 7.6 nmol/l and maximal number of binding sites (Bmax) of 0.94 and 0.95 pmol/mg protein. The rate constant of association, k1, was 3.7 X 10(8) l X mol-1 X min-1. Binding was slowly reversible with a t1/2 of 88 min. In competition experiments specific binding was most potently inhibited by (-)PIA, N6-cyclohexyladenosine (CHA), (-)HPIA and (-)IHPIA, followed by 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine. 1,3-Diethyl-8-phenylxanthine (DPX) and 8-phenyltheophylline were the most potent adenosine antagonists with Ki-values of 67 and 83 nmol/l, whereas the methylxanthines 3-isobutyl-1-methylxanthine, theophylline and caffeine had Ki-values between 1 and 21 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)