Mechanotransduction drives post ischemic revascularization through K(ATP) channel closure and production of reactive oxygen species.

AIMS: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a K(ATP) channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. RESULTS: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of K(ATP) channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1α) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of K(ATP) channel and in mice orally fed with a K(ATP) channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of K(ATP) channel or NOX2. Increased HIF-1α activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in K(ATP) channel and NOX2 null mice. Revascularization could be partially rescued in K(ATP) channel null mice by delivering VEGF into the hind limb. INNOVATION: This is the first report of a mechanosensitive ion channel (K(ATP) channel) initiating endothelial signaling that drives revascularization. CONCLUSION: The K(ATP) channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow.

Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes.

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4 degrees C, 1 h) of (125)I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of (125)I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of (125)I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

Identification of novel estrogen receptor alpha antagonists.

We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.

The ligand binding profiles of estrogen receptors alpha and beta are species dependent.

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.