Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+signaling in bovine vascular endothelial cells

Abstract

The role of glycolytically generated ATP in Ca2+/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca2+signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca2+concentration = 2 mM) to glycolytic inhibitors 2-deoxy-d-glucose (2-DG), pyruvate (pyr) + β-hydroxybutyrate (β-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca2+concentration ([Ca2+]i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca2+]i. The rise of [Ca2+]iwas characterized by a transient spike followed by a small sustained plateau of elevated [Ca2+]i. In the absence of extracellular Ca2+2-DG caused an increase in [Ca2+]i, suggesting that inhibition of glycolysis directly triggered release of Ca2+from intracellular endoplasmic reticulum (ER) Ca2+stores. The inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca2+response. Ca2+release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca2+]iwave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca2+]i. Propagating [Ca2+]iwaves were preceded by [Ca2+]ioscillations and small, highly localized elevations of [Ca2+]i(Ca2+puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca2+response, and vice versa during inhibition of CaMKII 2-DG-induced Ca2+release was attenuated. Similar results were obtained with pyr + β-HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg2+concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca2+release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP3R.