Inhibition of Turkey gizzard myosin light chain: Kinase activity by dihydropyridine calcium antagonists

Abstract

Drugs which block the influx of calcium (Ca 2−) across plasma membranes may additionally have direct effects upon smooth muscle contractile proteins. In a system of purified proteins comprised of calmodulin, turkey gizzard myosin light chains, and turkey gizzard myosin light chain kinase, the inhibition of myosin light chain phosphorylation by the dihydropyridine Ca 2+ antagonists felodipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(2,3-dichlorophenyl)-3,5-pyridine dicarboxylate] and nitrendipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate] was studied. In the presence of excess myosin light chain kinase, 50% inhibition of myosin light chain phosphorylation occurred at felodipine and nitrendipine concentrationsof 9.8 ± 1.1 × 10 −6 M and 5.6 ± 0.6 × 10 −5 M respectively. Inhibition of light chain kinase activity could not be overcome by increasing the free Ca 2+ concentration from 0.05 to 5.0 mM. Felodipine was unable to inhibit the activity of myosin light chain kinase rendered Ca 2+/calmodulin-independent by limited tryptic digestion. Using molecular sieve chromatography, nitrendipine was found to bind to calmodulin with an apparent dissociation constant ( K app) of 5.2 ± 0.3 × 10 −5 M, and this binding was Ca 2+ dependent. These data suggest that dihydropyridines inhibit the phosphorylation of smooth muscle myosin light chains in vitro by binding to Ca +/calmodulin and inhibiting the activation of myosin light chain kinase.