title: Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.
abstract: Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-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 (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x 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 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Evansville, IN 47712, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: McConnell
authors.forename: G W
authors.initials: GW
authors.lastname: White
authors.forename: J J
authors.initials: JJ
authors.lastname: Brokaw
authors.forename: B U
authors.initials: BU
authors.lastname: Raess
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D018871:Endoplasmic Reticulum, Smooth / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D007093:Imidazoles / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism / Q000493:pharmacokinetics; D013552:Swine
chemical_list: D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; C031938:calmidazolium; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium; D002118:Calcium