McGinnis K M, Wang K K, Gnegy M E
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Neurosci Lett. 2001 Mar 30;301(2):99-102. doi: 10.1016/s0304-3940(01)01629-9.
We previously demonstrated a loss in Ca(2+)/Calmodulin-dependent protein kinase (CaM kinase) activity in SH-SY5Y undergoing thapsigargin-mediated apoptosis. To extend that finding we report that CaM kinase inhibition potentiates thapsigargin-mediated cell death. CaM kinase inhibitor KN93 on its own exhibits little toxicity up to 10 mM, as measured by release of lactate dehydrogenase (LDH) into the culture medium. In SH-SY5Y cells pretreated with KN93 and the non-selective protein kinase inhibitor k252a and then treated with 2 mM thapsigargin, loss of viability is significantly greater than in cells treated with thapsigargin alone. Pretreatment with the pan-caspase inhibitor Z-D-DCB prevented the thapsigargin-mediated increase in LDH release. Furthermore, thapsigargin-induced caspase-3-like activation, demonstrated by poly(ADP)ribose polymerase cleavage and pro-caspase-3 processing, was elevated in the presence of KN93.
我们之前证明，在经历毒胡萝卜素介导的凋亡过程中的SH-SY5Y细胞中，钙/钙调蛋白依赖性蛋白激酶（CaM激酶）活性丧失。为了扩展这一发现，我们报告CaM激酶抑制会增强毒胡萝卜素介导的细胞死亡。钙调蛋白激酶抑制剂KN93在浓度高达10 mM时自身表现出很小的毒性，这通过乳酸脱氢酶（LDH）释放到培养基中的量来衡量。在用KN93和非选择性蛋白激酶抑制剂k252a预处理的SH-SY5Y细胞中，然后用2 mM毒胡萝卜素处理，细胞活力丧失明显大于仅用毒胡萝卜素处理的细胞。用泛半胱天冬酶抑制剂Z-D-DCB预处理可防止毒胡萝卜素介导的LDH释放增加。此外，在存在KN93的情况下，通过聚（ADP）核糖聚合酶裂解和前半胱天冬酶-3加工证明的毒胡萝卜素诱导的半胱天冬酶-3样激活增加。
