Interaction of the dihydropyridine calcium antagonist, CD-349, with calmodulin

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The characteristics of the binding of the 1,4-dihydropyridine Ca 2+ 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 Ca 2+/CaM-dependent enzyme, cyclic GMP (cGMP) phosphodiesterase (PDE), were investigated. CD-349 showed a Ca 2+-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 ( ic 50) of 2.4 μM, 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 ( K app) of CD-349 was 2.1 μM and the maximal number of binding sites ( B max) of CD-349 was 1.0 nmol/nmol CaM. In the presence of TFP, the K app and B max values of CD-349 binding to CaM were changed to 1.1 μM 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 Ca 2+/CaM-activated cGMP PDE activity. However, CaM did not competitively antagonize the CD-349-induced inhibition of the Ca 2+/CaM-activated PDE activity. In addition, the kinetic study showed that CD-349 inhibited both basal and Ca 2+/CaM-activated cGMP PDE activity, competitively with cGMP, with almost the same inhibition constant ( K i). These results suggest that CD-349 binds to CaM, with Ca 2+ dependency, at sites differing from those which bind to the CaM antagonist. The inhibitory activity of CD-349 on Ca 2+/CaM-dependent PDE does not seem to be due to a CaM antagonistic action.