Affiliation:
1. 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.
Abstract
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 · 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 2×10
−6
mol/L) with an EC
50
of 3.45±0.04×10
−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 (Ca
2+
+Mg
2+
)-ATPase activity in cultured porcine aortic endothelial cells.
Publisher
Ovid Technologies (Wolters Kluwer Health)
Subject
Cardiology and Cardiovascular Medicine,Physiology
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