Skip to main content
Fig. 4 | Molecular Medicine

Fig. 4

From: Caspase-Mediated Apoptosis in Neuronal Excitotoxicity Triggered by Nitric Oxide

Fig. 4

Caspase inhibitors prevent lethal execution steps downstream of NO-triggered and NMDA-R-mediated [Ca 2+ ] i increase

(A) CGC were loaded with fluo-3-AM and pretreated with solvent control (DMSO) or 2 µM MK801, 100 µM z-VAD-fmk, or 100 µM z-D-cbk, before they were challenged with GSNO. The increase of [Ca2+]i in time (f/fo fluorescence ratio) was followed by digital videoimaging. For comparison the insert shows the rapid [Ca2+]i increase following stimulation with SNOC or ONOO. Data are means ± SEM from 15 to 20 individually recorded neurons. (B) CGC [pretreated with z-D-cbk, MK801, or solvent control (DMSO)] were loaded with fura-2 4 hr after the challenge with 150 µM GSNO. [Ca2+]i were measured by ratiometric videoimaging and in situ calibration in 60–80 neurons from different preparations. (C) CGC were incubated with 1 µM colchicine in original culture medium or with low potassium medium (5 mM KCl) for 12 hr. Then they were incubated with 200 µM GSNO or PBS in the presence of MK801 for an additional 60 min, before caspase activity was determined. Samples were measured in the absence or presence of z-D-cbk or calpain-inhibitor m (CI-III) as indicated. Data for colchicine or low potassium treatments are presented as relative caspase activities compared with the following controls: colchicine without NO: 1430 U/mg = 100%; low potassium without NO: 2400 U/mg = 100%. (D) Serial dilutions of caspase inhibitors (DEVD = z-DEVD-fmk; D-cbk = z-D-cbk; YVAD = Ac-YVAD-cmk; VAD = z-VAD-fmk) or calpain-inhibitors (CI-I; CI-II, CI-III) were preincubated for 10 min with purified calpain. Calpain activity was measured by a kinetic assay based on the cleavage of suc-LLVY-amc. IC50 values were obtained by 4-parameter fit of the inhibition curves.

Back to article page