Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca signaling in bovine vascular endothelial cells

Aromolaran AS, Zima AV, Blatter LA. Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca signaling in bovine vascular endothelial cells. Am J Physiol Cell Physiol 293: C106–C118, 2007. First published March 7, 2007; doi:10.1152/ajpcell.00543.2006.—The role of glycolytically generated ATP in Ca /calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca concentration 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) -hydroxybutyrate ( -HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca concentration ([Ca ]i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca ]i. The rise of [Ca ]i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca ]i. In the absence of extracellular Ca 2-DG caused an increase in [Ca ]i, suggesting that inhibition of glycolysis directly triggered release of Ca from intracellular endoplasmic reticulum (ER) Ca stores. The inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93and 2-DG-induced Ca response. Ca release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca ]i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca ]i. Propagating [Ca ]i waves were preceded by [Ca ]i oscillations and small, highly localized elevations of [Ca ]i (Ca puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca response, and vice versa during inhibition of CaMKII 2-DG-induced Ca release was attenuated. Similar results were obtained with pyr -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP3R.