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

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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 simulated 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 EC50 of 3.45 ± 0.04X 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 (Ca2+ + Mg2+)-ATPase activity in cultured porcine aortic endothelial cells.

Original languageEnglish (US)
Pages (from-to)191-197
Number of pages7
JournalCirculation research
Volume86
Issue number2
DOIs
StatePublished - 2000

Keywords

  • (Ca +Mg)-ATPase
  • Aorta
  • Calmidazolium
  • Calmodulin
  • Endothelium

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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