Characterization of [ 3H]forskolin binding sites in the iris-ciliary body of the albino rabbit

Abstract

[ 3H]Forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that [ 3H]forskolin bound to a single population of high affinity sites. The K d and B max values were 8.7 ± 0.9 nM and 119.0 ± 30.9 fmol/mg prot. using membranes prepared from frozen tissue and 17.0 ± 6.2 nM and 184.4 ± 47.2 fmol/mg prot. using fresh tissue. The binding of [ 3H]forskolin was magnesium-dependent. The B max was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the modt potent inhibitor of [ 3H]forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the [ 3H]forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues.