flunarizine and 4-desmethoxyverapamil

Ca2+ channel blockers belonging to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes (Santos et al., J. Neurochem. 52, S49D, 1989). We now report that (-)-desmethoxyverapamil and flunarizine are the most potent inhibitors of the Ca(2+)-activated ATPase activity of synaptic plasma membranes, decreasing the Vmax by 41% and 37%, respectively, with no significant effects on the Km for Ca2+ (162.7 +/- 14.9 nM free [Ca2+]), while nitrendipine did not affect these parameters. Trifluoperazine was the most potent inhibitor of the Ca(2+)-activated ATPase of synaptic plasma membranes with an IC50 of 8-10 microM. To clarify whether the inhibitory effects of Ca2+ channel blockers and of trifluoperazine on the (Ca2+ + Mg2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125I-calmodulin to the membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer to nitrocellulose and autoradiography. The autoradiograms revealed Ca(2+)-dependent CaM binding proteins of about 140, 70 and 55 kDa. Trifluoperazine (30-40 microM) inhibited by 50-60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca2+ + Mg2+)-ATPase protein. Flunarizine and (-)-desmethoxyverapamil (100 microM) inhibited the 125I-calmodulin binding to the 140 kDa peptides by 100 and 90%, respectively, and they inhibited by 55 and 40%, respectively, the binding of 125I-calmodulin to the peptides in the 70-55 kDa range, whereas nitrendipine did not show any effect. The results suggest that the inhibitory effects of (-)-desmethoxyverapamil and flunarizine, as well as trifluoperazine, on the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.

Topics: Animals; Autoradiography; Binding Sites; Brain; Ca(2+) Mg(2+)-ATPase; Calcium Channel Blockers; Calmodulin; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Flunarizine; Iodine Radioisotopes; Nitrendipine; Sheep; Stereoisomerism; Synaptic Membranes; Trifluoperazine; Verapamil

The partition coefficients (Kp) of three prototype Ca2+ antagonists, nitrendipine, (-)-desmethoxyverapamil and flunarizine were determined in native synaptic plasma membranes (SPM) isolated from sheep brain cortex and in liposomes prepared with the total lipids extracted from the membranes. We found that at 25 degrees and at 5 x 10(-6) M drug concentration the Kp values of the drugs for native SPM are higher than those obtained for liposomes, and are of the order of 334 +/- 53, 257 +/- 36 and 23 X 10(3) for nitrendipine, (-)desmethoxyverapamil and flunarizine, respectively, whereas the Kp values in liposomes are 190 +/- 41, 118 +/- 10 and 6 x 10(3) for the same drugs. The results suggest that the presence of membrane proteins favors the incorporation of the drugs in the membranes. Furthermore, the Kp values of the three Ca2+ antagonists studied increase with temperature in native membranes, but not in liposomes. It is concluded that the physical partitioning in membranes of drugs which act on Ca2+ channels may play some role in the mechanism of interaction of these drugs with the Ca2+ channel proteins.

Topics: Animals; Brain; Calcium Channel Blockers; Flunarizine; Kinetics; Liposomes; Nitrendipine; Sheep; Synaptic Membranes; Verapamil