Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H₂O₂ (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.     

Proteomic analysis of the response of Arabidopsis chloroplast proteins to high light stress

Light is an essential environmental factor in the progression of plant growth and development but prolonged exposure to high levels of light stress can cause cellular damage and ultimately result in the death of the plant. Plants can respond defensively to this stress for a limited period and this involves changes to their gene expression profiles. Proteomic approaches were therefore applied to the study of the response to high light stress in the Arabidopsis thaliana plant species. Wild-type Arabidopsis was grown under normal light (100 micromol photons.m(-2).s(-1)) conditions and then subjected to high light (1000 micromol photons.m(-2).s(-1)) stress. Chloroplasts were then isolated from these plants and both soluble and insoluble proteins were extracted and subjected to two-dimensional (2-D) gel electrophoresis. The resolved proteins were subsequently identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and comparative database analysis. 64 protein spots, which were identified as candidate factors that responded to high light stress, were then selected for analysis and 52 of these were successfully identified using MALDI-TOF-MS analysis. 35 of the 52 identified proteins were found to decrease their expression levels during high light stress and a further 14 of the candidate proteins had upregulated expression levels under these conditions. Most of the proteins that were downregulated during high light stress are involved in photosynthesis pathways. However, many of the 14 upregulated proteins were identified as previously well-known high light stress-related proteins, such as heat shock proteins (HSPs), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD). Three novel proteins that were more highly expressed during periods of high light stress but had no clear functional relationship to these conditions, were also identified in this study.     

Functional and morphological characteristics of isolated pancreatic islets in tissue culture

Islets were isolated from the pancreas of female rats by using the collagenase technique. After culturing for 4 days, the islets were taken for measurement of insulin release biosynthesis as well as glucose utilization in subsequent short-time incubations. A low glucose concentration was insufficient to maintain a glucose-stimulated insulin release in vitro. A high glucose concentration had a protecting and restoring effect on the insulin release: ultrastructurally, such islets showed signs of an active biosynthesis in the electron micrograph. The enhancement of Mg++ in the culture medium resulted in an improvement of insulin storage in the islets, accompanied by a well-preserved action of glucose in a subsequent incubation.