Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced Ca2+ release.
Cyclic ADP-ribose (cADPR) is a naturally-occurring metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular Ca2+. A series of analogs modified at the 8-position of the adenine group were synthesized for the investigation of the relationship between the structure of the metabolite and its Ca(2+)-mobilizing activity. Substitution with an amino group at the 8-position of the adenine ring produced an antagonist. The 1H-NMR spectrum of 8-amino-cADPR showed characteristics of that of cADPR and confirmed the replacement of the 8-proton. By itself, 8-amino-cADPR (150 nM) did not induce Ca2+ release from sea-urchin-egg homogenates but totally blocked cADPR (135 nM) from doing so. The effect was reversible, since high concentrations of cADPR could overcome the inhibition. Addition of 8-amino-cADPR to egg homogenates during the cADPR-induced Ca2+ release blocked the release immediately, demonstrating the effectiveness of the antagonist. Measurements of [32P]cADPR binding to its microsomal binding site showed that 8-amino-cADPR was as effective as cADPR itself in competing for the binding site. In addition to blocking cADPR from releasing Ca2+, 8-amino-cADPR also inhibited cADPR from potentiating Ca(2+)-release induced by either divalent cations or by caffeine. Two other 8-substituted analogs were also synthesized. Both 8-Br- and 8-azido-cADPR were also antagonists, although with less potency than 8-amino-cADPR. These results show that alterations at the 8-position of the adenine group do not inhibit cADPR from binding to its receptor but do eliminate the ability of the metabolite to activate the Ca(2+)-release mechanism.
Walseth
T F
TF
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Lee
H C
HC
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000138:chemical synthesis*; D036563:Cyclic ADP-Ribose / Q000031:analogs & derivatives; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009865:Oocytes / Q000378:metabolism; D012617:Sea Urchins
D002121:Calcium Channel Blockers; D036563:Cyclic ADP-Ribose; C082175:8-aminoadenosine cyclic 3',5'-(hydrogen phosphate) 5'-ribofuranosyl ester; D000246:Adenosine Diphosphate Ribose; D000242:Cyclic AMP; D002118:Calcium