Interaction of the dihydropyridine calcium antagonist, CD-349, with calmodulin.
The characteristics of the binding of the 1,4-dihydropyridine Ca2+ antagonist, 2-nitratopropyl 3-nitratopropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine 3,5-dicarboxylate (CD-349), to calmodulin (CaM) and the effect of CD-349 on the Ca2+/CaM-dependent enzyme, cyclic GMP (cGMP) phosphodiesterase (PDE), were investigated. CD-349 showed a Ca2(+)-dependent binding to CaM, in equilibrium column binding studies. CD-349 inhibited the [3H]CD-349 binding to CaM, at a concentration producing a 50% inhibition (IC50) of 2.4 microM, whereas the CaM antagonist, trifluoperazine hydrochloride (TFP), stimulated the [3H]CD-349 binding to CaM. Scatchard plot analysis of the binding of CD-349 to CaM revealed that the apparent dissociation constant (Kapp) of CD-349 was 2.1 microM and the maximal number of binding sites (Bmax) of CD-349 was 1.0 nmol/nmol CaM. In the presence of TFP, the Kapp and Bmax values of CD-349 binding to CaM were changed to 1.1 microM and 1.5 nmol/nmol CaM respectively. Although the CaM antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and TFP, decreased and increased, respectively, the fluorescence intensity caused by 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS)-CaM binding, CD-349 only slightly decreased the fluorescence of TNS bound CaM. CD-349 inhibited both basal and Ca2+/CaM-activated cGMP PDE activity. However, CaM did not competitively antagonize the CD-349-induced inhibition of the Ca2+/CaM-activated PDE activity. In addition, the kinetic study showed that CD-349 inhibited both basal and Ca2+/CaM-activated cGMP PDE activity, competitively with cGMP, with almost the same inhibition constant (Ki). These results suggest that CD-349 binds to CaM, with Ca2+ dependency, at sites differing from those which bind to the CaM antagonist. The inhibitory activity of CD-349 on Ca2+/CaM-dependent PDE does not seem to be due to a CaM antagonistic action.
Tanaka
M
M
Research Center, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.
Muramatsu
M
M
Aihara
H
H
Otomo
S
S
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism* / Q000494:pharmacology; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects
D002121:Calcium Channel Blockers; D002147:Calmodulin; D004095:Dihydropyridines; C054179:CD 349; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases