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Figure 1


Fig. 1. NO activates calpain in osteoclasts, antagonists of NO or cGMP inactivate it. (A) Fluorescent calpain substrate assays in living NO-inhibited or NO-activated osteoclasts. Cells were incubated 20 minutes in 50 µM of the cell-permeable calpain substrate BOC. Quenching BOC is separated from the fluorescent coumarin when proteinase activity cleaves the peptide linker (Leu-Met), increasing fluorescence intensity. Untreated cells are compared with cells incubated with the NO donor SNP (100 µM), the activating cGMP analog 8-pCPT-cGMP (100 µM), the cGMP-blocking analog Rp-cGMPS (50 µM) and the inhibitor of NO synthase L-NMMA (1 µM). Results from four experiments, each measuring a minimum of 16 cells, summarized as the mean ± s.d. of each experiment, normalized to untreated controls. The NO and cGMP agonists increased calpain activity relative to untreated controls (*P<0.05), and relative to the inhibitors Rp-cGMPS and L-NMMA (P<0.01). Inhibitors reduced substrate degradation relative to untreated control, but the difference was significant only for Rp-cGMPS ({dagger}P<0.05). Note that, in the presence of Rp-cGMPS, the change in calpain activity after NO addition is small and not significant (fifth versus fourth bar). (B) Images of BOC fluorescence in cells with key treatments. SNP (right) increased fluorescence in essentially all of the cells with respect to control (left). Inhibitors such as L-NMMA (middle) reduce activity relative to untreated control, but the difference varies from experiment to experiment due to the variability in autocrine NO production in untreated cells. This is reflected also in the variability of response of osteoclasts to Rp-cGMPS and L-NMMA shown in A. (C) Calpain activity is suppressed by knockdown of VASP or PKG1. BOC fluorescence assays were carried out as in A but cells had been transfected with scrambled control siRNA or siRNAs targeting VASP and PKG1. The cells had been transfected with Cy3-labeled siRNAs 72 hours prior to the assay, and treated with 100 µM 8-pCPT-cGMP for 60 minutes prior to the assay. The assay scored BOC fluorescence over background in cells labeled with Cy3 (red). PKG1 suppression and VASP suppression reduced cGMP-stimulated calpain activity relative to the control (*P<0.05, in both cases; n=6, mean ± s.e.m.). Western blots for knockdown efficacy are shown in Fig. 6.