Role of calmodulin in thyroid hormone stimulation in vitro of human erythrocyte Ca2+-ATPase activity.
Because human erythrocyte membrane Ca2+-ATPase is a calmodulin-dependent enzyme, and because physiological levels of thyroid hormone stimulate this enzyme system in vitro, we have studied the role of calmodulin in this model of extranuclear thyroid hormone action. Ca2+-ATPase activity in the absence of thyroid hormone ("basal activity") was increased by inclusion in the preassay incubation mixture of purified calmodulin or hypothyroid erythrocyte hemolysate that contained calmodulin (39 micrograms calmodulin/ml packed cells, determined by radioimmunoassay); addition of L-thyroxine or 3,5,3'-triiodo-L-thyronine (10(-10)M) significantly enhanced (P less than 0.001) enzyme activity in the presence of calmodulin or hemolysate. The stimulatory effects of thyroid hormone, calmodulin, and hemolysate were additive. At 5-10 microM, trifluoperazine, an antagonist of calmodulin, inhibited thyroid hormone stimulation of Ca2+-ATPase activity. Higher concentrations of trifluoperazine (50-100 microM) inhibited basal and hormone-stimulated enzyme activity, with or without added calmodulin. Anti-calmodulin antibody (10-50 micrograms antibody/mg membrane protein) inhibited basal, calmodulin-stimulated and thyroid hormone-stimulated Ca2+-ATPase activity. Membrane preparations were shown by radioimmunoassay to contain residual endogenous calmodulin (0.27 +/- 0.02 micrograms/mg membrane protein). The latter accounts for the effect of trifluoperazine and calmodulin antibody on membrane Ca2+-ATPase activity in the absence of added purified calmodulin. These results support the conclusion that the in vitro action of physiological levels of iodothyronines on human erythrocyte Ca2+-ATPase activity requires the presence of calmodulin.
D000906:Antibodies; D002135:Calcium-Binding Proteins / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000276:immunology / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D013963:Thyroid Hormones / Q000494:pharmacology*; D014268:Trifluoperazine / Q000494:pharmacology
D000906:Antibodies; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D013963:Thyroid Hormones; D014268:Trifluoperazine; D000252:Calcium-Transporting ATPases