Modulation of intracellular Ca2+release and capacitative Ca2+entry by CaMKII inhibitors in bovine vascular endothelial cells

Abstract

The effects of inhibitors of CaMKII on intracellular Ca2+signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca2+concentration ([Ca2+]i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca2+]itransient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca2+resulted in a dose-dependent increase of [Ca2+]iconsisting of a rapid and transient Ca2+spike followed by a small sustained plateau phase of elevated [Ca2+]i. Exposure to KN-93 in the absence of extracellular Ca2+caused a transient rise of [Ca2+]i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca2+from intracellular endoplasmic reticulum (ER) Ca2+stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca2+largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca2+response, suggesting that exposure to KN-93 affects Ca2+release from an IP3-sensitive store. Depletion of Ca2+stores by exposure to ATP or to the ER Ca2+pump inhibitor thapsigargin triggered robust capacitative Ca2+entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca2+handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca2+from intracellular stores and activation of CCE.