Zinc regulates reactive oxygen species generation in platelets.

Vascular complications resulting from atherosclerosis development are a major cause of death. Reactive oxygen species (ROS) are produced by platelets during activation, and have been demonstrated to positively regulate platelet activatory responses. Zn2+ is also an important hemostatic cofactor in platelets, acting both as a platelet agonist and second messenger. Whilst the effect of Zn2+-dependent signaling mechanisms on ROS production in nucleated cells has been demonstrated, comparable roles in platelets have yet to be investigated. In this study we investigated the relationship between fluctuations in cytosolic Zn2 [Zn2+]i and platelet ROS production. Agonist-evoked ROS production, GSH levels and GPx activity are abrogated in platelets treated with the Zn2+-chelator, TPEN. Conversely, increasing platelet [Zn2+]i using Zn2+ ionophores potentiated ROS generation and decreased GSH levels and GPx activity. Zn2+-dependent ROS production was sensitive to pretreatment with DPI or mitoTEMPO, NADPH oxidase and mitochondria inhibitors respectively. Increasing [Zn2+]i resulted in increases of Erk1/2 and JNK phosphorylation. Our data are consistent with a functional association between [Zn2+]i and ROS production in platelets that could influence thrombus formation in a clinical context.

Reactive oxygen species: physiological roles in the regulation of vascular cells.

Reactive oxygen species (ROS) are now appreciated to play several important roles in a number of biological processes and regulate cell physiology and function. ROS are a heterogeneous chemical class that includes radicals, such as superoxide ion (O2(•-)), hydroxyl radical (OH(•)) and nitric oxide (NO(•)), and non-radicals, such as hydrogen peroxide (H2O2), singlet oxygen ((1)O2), hypochlorous acid (HOCl), and peroxynitrite (NO3 (-)). In the cardiovascular system, besides playing a critical role in the development and progression of vasculopathies and other important pathologies such as congestive heart failure, atherosclerosis and thrombosis, ROS also regulate physiological processes. Evidence from a wealth of cardiovascular research studies suggests that ROS act as second messengers and play an essential role in vascular homeostasis by influencing discrete signal transduction pathways in various systems and cell types. They are produced throughout the vascular system, regulate differentiation and contractility of vascular smooth muscle cells, control vascular endothelial cell proliferation and migration, mediate platelet activation and haemostasis, and significantly contribute to the immune response. Our understanding of ROS chemistry and cell biology has evolved to the point of realizing that different ROS have distinct and important roles in cardiovascular physiology. This review will outline sources, functions and molecular mechanisms of action of different ROS in the cardiovascular system and will describe their emerging role in healthy cardiovascular physiology and homeostasis.

Sonographic evaluation of the lower uterine segment thickness in women with a single previous Cesarean section.

AIM: The aim of this paper was to evaluate the lower uterine segment (LUS) thickness through transvaginal sonography in late preterm and full term pregnancies with a single previous Cesarean section, to correlate the obtained LUS measurements with intraoperative observations, and to identify a predictive cut-off value in order to select the best candidates for a vaginal birth after Cesarean delivery (VBAC). METHODS: Two hundred and fourteen women with a single previous Cesarean section who had an ultrasound measurement of the LUS thickness (stratified in S1, S2 and S3) in pregnancy were enrolled. The outcome of interest was the visual finding of a thin uterine scar at the time of the iterative Cesarean section. Linear regression was used to correlate the LUS thickness with gestational age (GA). A ROC curve has been used to determine the detection rate (DR) and the risk of each actual value of LUS thickness versus a thin uterine scar (outcome of interest). RESULTS: The LUS thickness was correlated with the gestational age (R2=0.034, P-value =0.005). The DR as estimated by ROC curves to detect a translucent lower uterine segment (S3) was 94.1% at a false positive rate (FPR) of 20%. The correspondent cut-off value was 1.8 mm. Finally a likelihood ratio (LR) of observing S3 was estimated. At the quoted cut-off of 1.8 mm the LR was 3. As demonstrated, for a segment of 1 mm the LR was instead about 13. CONCLUSION: The obtained values lead us to the conclusion that a thickness less than 1.8 mm can be reasonably considered a valid cut-off value to identify patients with a higher risk of thin uterine scar.

Vaginal delivery rate in post-term pregnancies with one versus more than one dinoprostone gel administrations: an observational study.

AIM: The study aims to calculate the probability of a successful vaginal delivery in post-term low-risk women by using a set of predictors (maternal, fetal, and ultrasonographic) according to the number of dinoprostone gel applications. METHODS: This was an observational study of a cohort of 174 low-risk post-term singleton pregnancies. Parity, cervical status and length, and amniotic fluid volume (AFI) were evaluated immediately before prostaglandin gel induction at the Department of Obstetrics and Gynecology, University of Bologna, Bologna, Italy between January 2010 and October 2011. RESULTS: A consistent difference in vaginal delivery rates was observed for women who had one gel administration (Group 1) versus those who received either two or three gel administrations (Group 2): 77.5% at 24 hours and 97.4% at 48 hours vs. 27% at 24 hours and 54% at 48 hours (P <0.001 for both the comparisons) respectively. The predictors of a vaginal delivery were cervix dilatation and short cervix for Group 1, lower AFI for Group 2, and parity for both groups. CONCLUSION: Women who require more than one gel administration have a lower rate of vaginal delivery at 24 and 48 hours. Maternal, fetal, and ultrasonographic parameters can predict a successful vaginal delivery.

The novel NOX inhibitor 2-acetylphenothiazine impairs collagen-dependent thrombus formation in a GPVI-dependent manner.

BACKGROUND AND PURPOSE: NADPH oxidases (NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2-acetylphenothiazine (2-APT) on human platelet functional responses and intracellular signaling pathways. EXPERIMENTAL APPROACH: The generation of superoxide ions was assessed by single cell imaging on adhering platelets using dihydroethidium (DHE), while other reactive oxygen species (ROS) were detected with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)-DCFDA). Whole blood thrombus formation, washed platelet aggregation, integrin αIIbß3 inside-out signalling, Syk phosphorylation and PKC activation were analysed to understand the functional consequences of NOX inhibition by 2-APT in platelets. KEY RESULTS: Superoxide ion generation stimulated by platelet adhesion on collagen and fibrinogen was significantly inhibited by 2-APT in concentration-dependent manner (IC(50) = 306 nM and 227 nM, respectively), whereas cumulative ROS accumulation was not affected by this pharmacological agent. 2-APT also abolished collagen-dependent whole blood thrombus formation and washed platelet aggregation in response to collagen but not thrombin. The activation of integrin αIIbß3 and PKC in response to the GPVI-specific agonist collagen-related peptide (CRP) was significantly reduced, whereas the same responses to thrombin were not significantly affected by 2-APT. Finally, Syk activation in response to collagen but not thrombin was inhibited by 2-APT. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that 2-APT attenuates GPVI-specific signalling and is a novel inhibitor of collagen-induced platelet responses. Therefore, NOXs could represent a novel target for the discovery of anti-thrombotic drugs.

Role of Ena/VASP proteins in homeostasis and disease.

The actin cytoskeleton is required for many important processes during embryonic development. In later stages of life, important homeostatic processes depend on the actin cytoskeleton, such as immune response, haemostasis and blood vessel preservation. Therefore, the function of the actin cytoskeleton must be tightly regulated, and aberrant regulation may cause disease. A growing number of proteins have been described to bind and regulate the actin cytoskeleton. Amongst them, Ena/VASP proteins function as anti-capping proteins, thereby directly modulating the actin ultrastructure. Ena/VASP function is regulated by their recruitment into protein complexes downstream of plasma membrane receptors and by phosphorylation. As regulators of the actin ultrastructure, Ena/VASP proteins are involved in crucial cellular functions, such as shape change, adhesion, migration and cell-cell interaction and hence are important targets for therapeutic intervention. In this chapter, we will first describe the structure, function and regulation of Ena/VASP proteins. Then, we will review the involvement of Ena/VASP proteins in the development of human diseases. Growing evidence links Ena/VASP proteins to important human diseases, such as thrombosis, cancer, arteriosclerosis, cardiomyopathy and nephritis. Finally, present and future perspectives for the development of therapeutic molecules interfering with Ena/VASP-mediated protein-protein interactions are presented.

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent.

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.

Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines.

A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel. The analysis of known blockers of calcium-activated potassium channels revealed the highest potency for apamin (IC(50)=13.2 nM). This result was confirmed by direct recordings of SK3 currents using the whole-cell patch-clamp technique. Tricyclic antidepressants such as desipramine, imipramine and nortriptyline as well as phenothiazines such as fluphenazine, promethazine, chlorpromazine and trifluoperazine blocked the hSK3 channel with micromolar potencies. These compounds also displaced [(125)I]-apamin binding to the hSK3 channel thus suggesting direct and competitive channel blocking activity. Since these compounds share a common three-ring molecular core structure, this feature seems to be important for channel blocking activity. The serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A were able to abolish channel activation with nanomolar potencies, but did not displace [(125)I]-apamin binding. Thus, phosphorylation of hSK3 or an accessory channel subunit seems to be involved in its modulation.

Annexin VI isoforms are differentially expressed in mammalian tissues.

Purified annexin VI migrates as a closely spaced doublet when separated by SDS-PAGE. Immunolocalization of annexin VI in heart demonstrates staining at different defined subcellular compartments. Moss et al. identified two cDNAs, one having an insert of 18 bases encoding VAAEIL at the beginning of repeat domain seven. We have identified the splicing site of the murine annexin VI gene. It contains a single small exon of 18 bases. PCR amplification of reverse transcribed (RT) mRNA demonstrates that, in all tissues tested, the mRNA isoform containing the insert is predominant. Site-directed antibody was produced and affinity purified against peptides reflecting the insert and deletion sequences. The steady-state isoform ratio of the annexin VI protein is consistent with the RT-PCR data. Chromatographic experiments demonstrate that the annexin VI protein isoforms have biochemical differences. These differences may target the individual isoforms to unique cellular compartments or alter functional properties.

S-100 protein binds to annexin II and p11, the heavy and light chains of calpactin I.

S-100 protein, a dimeric, Ca(2+)-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p11 (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-p11(2) (calpactin I) in a Ca(2+)-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (Kd approx. 0.4 microM) and with the lowest affinity to calpactin I (Kd approx. 10 microM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p11 in regulating the activities of annexin II in cells which do not express p11 and/or act synergistically with p11 in cells expressing both p11 and S-100. The binding of S-100 protein to p11 could reflect the natural tendency of S-100 subunits and p11 to dimerize. Chimeric p11-S-100 alpha and p11-S-100-beta proteins could therefore form in a Ca(2+)-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.

Novel isoforms of CaBP 33/37 (annexin V) from mammalian brain: structural and phosphorylation differences that suggest distinct biological roles.

Two calcium-dependent phospholipid- and membrane-binding proteins have been purified from bovine brain. These are termed CaBP33 and CaBP37. Complete sequence analysis has revealed that these two proteins are isoforms of annexin V. Despite an apparent difference of 4 kDa between the two proteins on SDS-PAGE, only two amino-acid substitutions were found. These are, in CaBP33, Ser-36 and Lys-125 and in CaBP37, Thr-36 and Glu-125. This corresponds to a mass difference of 15 Da. This was confirmed by electrospray mass spectrometric analysis. Both isoforms can be phosphorylated substoichiometrically in vitro by protein kinase C at residue Thr-22.

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.

'Neuron-specific' protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state.

Protein gene product 9.5 (PGP 9.5), which in the normal nervous system is restricted to certain neurons, has been detected in two glioma cell lines, rat C6 and human GL15, by immunoblotting and immunocytochemistry. Its expression in these cells depends on the cellular growth state, being maximal between the first and second post-plating day. Only a faint PGP 9.5 immunoreactivity can be observed in glioma cells after the eleventh post-plating day, i.e. about one week after confluency has been reached. The present results suggest that PGP 9.5 in cultured glial cells is maximally expressed during the growth phase and that the protein could play a role during brain development in glial cells, in reactive gliosis, or in tumorigenesis of the glial lineage.

Immunohistochemical localization of annexin V (CaBP33) in rat organs.

We investigated the cellular distribution of annexin V (CaBP33) in rat tissues by immunohistochemistry. Several cell types were shown to express the protein. Glial cells in the cerebellum and in the optic nerve, the corneal epithelium, the posterior epithelium in the iris, chondrocytes, skeletal muscle cells and cardiomyocytes, the capillary endothelial cells in many organs, the muscularis mucosae and the muscular layer in the intestinal tract, hepatocytes, Müller cells in the retina, the lens fibers, Sertoli and Leydig cells in the testis, and smooth muscle cells in the epididymis and bronchi displayed intense immunostaining. In the adrenal gland, only the cortex showed immunoreaction product. In the kidney, no apparent staining of renal cells was observed, whereas endothelial cells of peritubular capillaries were stained. In the heart, annexin V was found associated exclusively with the sarcolemma and intercalated discs, as opposed to the diffuse distribution of the protein in skeletal muscle cells. In the spleen, only reticular elements in the white pulp and endothelial cells in the red pulp appeared to be immunostained. The present data complement the biochemical work thus far done on annexin V and suggest that the protein is neither restricted to secretory cells nor exclusively related to exocytotic events in secretory cells.

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V).

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 microM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.

Interaction of two brain annexins, CaBP33 and CaBP37, with membrane-skeleton proteins.

CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X-100-resistant fraction (cytoskeleton) from bovine brain membranes in a Ca2(+)-dependent way in vitro. The binding is saturable with respect to the CaBP33-CaBP37 concentration, half-maximal binding occurring at approximately 15 micrograms of the CaBP33-CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca2+ concentration is biphasic, with half-maximal binding at approximately 50 microM and approximately 400 microM free Ca2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low Mr polypeptides (10-20 kDa) in the crude cytoskeleton preparation, with formation of an 85-90 kDa complex as investigated in cross-linking experiments. No binding of the CaBP33-CaBP37 mixture to either G- or F-actin has been observed. Identification of the CaBP33-CaBP37-binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.

Two novel brain proteins, CaBP33 and CaBP37, are calcium-dependent phospholipid- and membrane-binding proteins.

Two acidic Ca2(+)-binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat-sensitivity, electrophoretic mobility, and Ca2(+)-dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane-bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KCl. However, apparent Ca2(+)-independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane-bound CaBP33 and CaBP37 could be solubilized with Triton X-100, indicating that brain membranes normally contain the two proteins as intrinsic components.