Localized activation of m-calpain in human umbilical vein endothelial cells upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found the appearance of the localized cytoplasmic ionized Ca concentration ([Ca2+](i)) rise before bleb formation at the same loci of human umbilical vein endothelial cell (HUVEC) upon hypoxia, the mode of [Ca2+](i)-rise-induced cytoskeletal alteration remains ill-defined. The aim of this study is to clarify the mechanisms causing bleb formation after localized [Ca2+](i) rise. We studied the activation of m-calpain associated with the alteration of cytoskeleton-related proteins, F-actin, mu -actin, or ezrin by employing specific antibodies in conjunction with a confocal laser scanning microscopy (CLSM). Specific antibodies against 80-kDa-preactivated and 78-kDa-activated m-calpain clearly demonstrated redistribution of 80-kDa m-calpain followed by autoproteolytic activation of m-calpain to the 78-kDa form at the same loci of [Ca2+](i) rise in hypoxia-treated HUVECs, which was associated with the decrease of ezrin and the localized appearance of beta -actin at the same loci. In conclusion, hypoxia-induced localized [Ca2+](i) rise causes bleb formation at the same loci through m-calpain-catalyzed destruction of cross-linking between plasma membrane and actin filaments. (C) 2001 Elsevier Science Ltd. All rights reserved.