Oxysterol-induced apoptosis of smooth muscle cells is under the control of a soluble adenylyl cyclase.

AIMS: Apoptosis of vascular smooth muscle cells (VSMC) in advanced atherosclerotic plaques is an important cause of plaque instability. Oxysterols have been suggested as important inducers of apoptosis in VSMC, but the precise mechanism is still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). METHODS AND RESULTS: VSMC derived from rat aorta were treated with either 25-hydroxycholesterol or 7-ketocholesterol for 24 h. Apoptosis was detected by TUNEL staining and caspases cleavage. Oxysterols treatment led to the activation of the mitochondrial pathway of apoptosis (cytochrome c release and caspase-9 cleavage) and mitochondrial ROS formation, which were suppressed by the pharmacological inhibition or knockdown of sAC. Scavenging ROS with N-acetyl-l-cysteine prevented oxysterol-induced apoptosis. Analyses of the downstream pathway suggest that protein kinase A (PKA)-dependent phosphorylation and the mitochondrial translocation of the pro-apoptotic protein Bax is a key link between sAC and oxysterol-induced ROS formation and apoptosis. To distinguish between intra-mitochondrial and extra-mitochondrial/cytosolic sAC pools, sAC was overexpressed in mitochondria or in the cytosol. sAC expression in the cytosol, but not in mitochondria, significantly promoted apoptosis and ROS formation during oxysterol treatment. CONCLUSION: These results suggest that the sAC/PKA axis plays a key role in the oxysterol-induced apoptosis of VSMC by controlling mitochondrial Bax translocation and ROS formation and that cytosolic sAC, rather than the mitochondrial pool, is involved in the apoptotic mechanism.

Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury.

Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.

The mechanisms of energy crisis in human astrocytes after subarachnoid hemorrhage.

BACKGROUND: Calcium (Ca2+) is a cofactor of multiple cellular processes. The mechanisms that lead to elevated cytosolic Ca2+ concentration are unclear. OBJECTIVE: To illuminate how bloody cerebrospinal fluid (bCSF) from patients with intraventricular hemorrhage causes cell death of cultured human astrocytes. METHODS: Cultured astrocytes were incubated with bCSF. In control experiments, native CSF was used. Cytosolic Ca2+ concentration was measured by fura-2 fluorescence. Apoptosis and necrosis were evaluated by staining with Hoechst-3342 and propidium iodide. RESULTS: Incubation of astrocytes with bCSF provoked a steep Ca2+ concentration peak that was followed by a slow Ca2+ rise during the observation period of 50 minutes. Necrosis, but not apoptosis, was induced. Blockade of ATP-sensitive P2 receptors with suramin inhibited the bCSF-induced initial Ca2+ peak and necrosis. Blockade of P1 receptors with 8-phenyltheophylline or of N-methyl-D-aspartate receptors with D(-)-2-amino-5-phosphopentanoic acid had no significant effect. Preincubation with xestospongin D, a blocker of inositol 1,4,5-trisphosphate receptors, prevented the initial Ca2+ rise and reduced the rate of necrosis. Preemptying of the endoplasmic reticulum with thapsigargin protected astrocytes from the bCSF-induced Ca2+ peak. Inhibition of mitochondrial permeability transition pores opening with cyclosporin A reduced the rate of astrocytic necrosis significantly, although it did not influence the initial Ca peak. CONCLUSION: bCSF elicits a steep, transient Ca rise when administered to human astrocytes by activation of ATP-sensitive P2 receptors and subsequent inositol 1,4,5-trisphosphate-dependent Ca release from endoplasmic reticulum. This massive Ca overload leads to subsequent mitochondrial permeability transition pores opening and necrosis of the cells.