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(Circulation Research. 2000;86:191.)
© 2000 American Heart Association, Inc.

Cellular Biology

Pharmacological and Immunohistochemical Characterization of Calmodulin-Stimulated (Ca²⁺+Mg²⁺)-ATPase in Cultured Porcine Aortic Endothelial Cells

Elizabeth J. McConnell, Gary W. White, James J. Brokaw, Beat U. Raess

From the Departments of Pharmacology and Toxicology (E.J.M., B.U.R.) and Anatomy and Cell Biology (G.W.W., J.J.B.), Indiana University School of Medicine, Evansville, Ind.

Correspondence to B. Raess, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 8600 University Blvd, Evansville, IN 47712. E-mail braess{at}iupui.edu

Abstract—Plasma membrane (Ca²⁺+Mg²⁺)-ATPase and Ca²⁺ transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca²⁺ signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca²⁺+Mg²⁺)-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca²⁺- and/or Mg²⁺-activated ecto–5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10^-9 to 10^-6 mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca²⁺+Mg²⁺)-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti–(Ca²⁺+Mg²⁺)-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca²⁺ pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca²⁺+Mg²⁺)-ATPase, 10^-5 mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca²⁺+Mg²⁺)-ATPase activity from 143.0 to 78.15 nmol P_i/mg protein · min^–1. This calmidazolium-reduced activity could then be stimulated 113.1±0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10^-7 to 2x10^-6 mol/L) with an EC₅₀ of 3.45±0.04x10^-7 mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca²⁺+Mg²⁺)-ATPase activity in cultured porcine aortic endothelial cells.

Key Words: endothelium • aorta • (Ca²⁺+Mg²⁺)-ATPase • calmodulin • calmidazolium