Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase

A Laurenza, JD Robbins and KB Seamon

Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2- aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3- (3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP- Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP- Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP- Fsk. The time dependence and concentration dependence for the BrAcFsk- induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP- Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3- thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4- fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 41, Issue 2, pp. 360-368, 02/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics

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