Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca2++Mg2+)-ATPase in cultured porcine aortic endothelial cells

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) moI/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 approximate to 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.