Rapamycin inhibits hydrogen peroxide-induced loss of vascular contractility

Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, has been shown to extend the life span of mice, and oxidative stress plays critical roles in vascular aging involving loss of compliance of arteries. We examined, therefore, whether rapamycin has protective effects on the inhibition of vascular contractility by hydrogen peroxide (H2O2). Prolonged (3 h) exposure to H2O2 induced complete loss of contraction of mouse aortic rings and mesenteric (resistance) arteries to either KCl or phenylephrine, which was attenuated by pretreatment with rapamycin. H2O2-induced loss of contractility was unaffected by treatment with actinomycin D or cycloheximide, inhibitors of gene transcription and protein synthesis, respectively. Western blot analysis showed that there was no increase in phosphorylation of S6 kinase 1 (S6K) or factor 4E binding protein 1 (4EBP1) in response to H2O2 treatment, suggesting involvement of the mTOR complex-2 (mTORC2) rather than mTORC1. H2O2 treatment inhibited phosphorylation of the 20-kDa regulatory light chains of myosin (LC20), which was partially blocked by rapamycin treatment. Interestingly, the calcineurin inhibitors cyclosporine A and FK506 were found to mimic the rapamycin effect, and rapamycin inhibited calcineurin activation induced by H2O2. We conclude that rapamycin inhibits H2O2-induced loss of vascular contractility, likely through an mTORC2-calcineurin pathway.

Cyclophilin B Binding to Platelets Supports Calcium-Dependent Adhesion to Collagen

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 ± 3 nmol/L and the number of sites estimated at 960 ± 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca2+ and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca2+ channel.

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.