Effects of microtubular inhibitors on plasma membrane calmodulin-dependent Ca2+-transport ATPase
Gietzen, K.; Wüthrich, A.; Bader, H.
Molecular Pharmacology 22(2): 413-420
1982
ISSN/ISBN: 0026-895X PMID: 6216398 Document Number: 190327
Several microtubular inhibitors were examined for their effects on human red blood cell calmodulin-dependent Ca2+-transport ATPase. The Vinca alkaloids vinblastine, vindesine and vincristine were inhibitors of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase, with IC50 values of 35, 100 and 220 .mu.M, respectively. However, in the concentration range of 1-10 .mu.M all 3 agents inhibited 10-14% of the calmodulin-dependent ATPase activity. The Vinca alkaloids were far less effective in antagonizing Ca2+-transport ATPase activity in the absence of added calmodulin. In contrast, the potent microtubular inhibitors, nocodazole and colchicine, failed to inhibit significantly the calmodulin-dependent Ca2+-transport ATPase. The concentration of vinblastine required for 50% inhibition of the calmodulin-dependent fraction of Ca2+ transport into inside-out vesicles of red blood cells was very similar to that found for the Ca2+-ATPase. Kinetic analysis revealed that the activation of Ca2+-transport ATPase induced by calmodulin is inhibited by vinblastine according to a mixed-type mechanism, although the mechanism is mostly of a competitive nature. The mechanism of action of vinblastine on calmodulin-dependent Ca2+-transport ATPase was further elucidated by means of binding studies with radioactively labeled vinblastine. Vinblastine was found to bind to calmodulin as well as to Ca2+-transport ATPase of red blood cells. Binding of vinblastine to calmodulin required the presence of Ca2+, whereas binding to Ca2+-transport ATPase occurred Ca2+-independently. Calmodulin has 2 classes of vinblastine binding sites: one site with a high affinity for vinblastine (KD = 2 .mu.M) and one additional site with a lower affinity for the drug (KD = 10 .mu.M). With respect to other ATPases of the red blood cell membrane, the action of vinblastine on Ca2+-transport ATPase seems to be specific since Mg2+-ATPase and (Na+ + K+)-transport ATPase were hardly affected at concentrations up to 300 .mu.M. The antagonism of calmodulin effects by Vinca alkaloids may be of general importance for all calmodulin-regulated parameters.