Nakano Tomoyasu, Watanabe Hiroshi, Ozeki Mariko, Asai Masayoshi, Katoh Hideki, Satoh Hiroshi, Hayashi Hideharu
Department of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
Cardiovasc Res. 2006 Mar 1;69(4):908-15. doi: 10.1016/j.cardiores.2005.11.023. Epub 2005 Dec 27.
Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells.
Cytosolic Ca(2+) concentration (Ca(2+)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.
Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased Ca(2+), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased Ca(2+) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis.
Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.
内皮细胞凋亡被认为是动脉粥样硬化发展的起始步骤。最近的研究表明，内质网（ER）钙含量的减少在凋亡过程中起重要作用。半胱天冬酶 - 12是内质网应激诱导凋亡的关键信号。然而，尚不清楚内质网钙的减少是否与内皮细胞中的半胱天冬酶 - 12信号传导有关。在此，我们研究了钙信号传导与半胱天冬酶 - 12切割在内皮细胞凋亡中的相互作用。
使用fura - 2/AM测量原代猪主动脉内皮细胞的胞质钙浓度（Ca(2 +)）。通过DNA片段化评估凋亡，并使用蛋白质印迹技术检测半胱天冬酶 - 12的切割情况。
内质网钙ATP酶抑制剂毒胡萝卜素（5 microM）可耗尽内质网钙含量，增加Ca(2 +)，切割半胱天冬酶 - 12，并诱导凋亡。缓激肽（10 nM）也可增加Ca(2 +)，但不切割半胱天冬酶 - 12或诱导凋亡。然而，当细胞内钙用BAPTA/AM（100 microM）螯合时，缓激肽导致内质网钙耗竭和凋亡，但不伴随半胱天冬酶 - 12切割。非选择性半胱天冬酶抑制剂z - VAD.fmk（100 microM）可抑制毒胡萝卜素刺激的凋亡和半胱天冬酶 - 12切割，而钙蛋白酶抑制剂MDL 28170（120 microM）可抑制半胱天冬酶 - 12切割，但不抑制凋亡。
因此，细胞内钙浓度的增加不足以诱导内皮细胞凋亡，内质网钙耗竭似乎独立于半胱天冬酶 - 12诱导凋亡。
