Blood bicarbonate, which is in direct contact with the endothelium, can become elevated during respiratory diseases or following infusion (i.e to correct acidosis associated with lung protective mechanical ventilation strategies), yet its effect on the endotheliul are unknown. Na+-bicarbonate cotransporters (NBC) transport bicarbonate into cells. In the cytosol, bicarbonate can activate cytosolic adenylyl cyclase isoform 10, AC10, which generates cytosolic cAMP and disrupts the pulmonary endothelial barrier. The goal of this proposed research plan is to understand how the NBC is regulated and the mechanism of AC10 induced permeability in the setting of acute lung injury (ALI). It has been observed that while transmembrane AC generates juxtamembrane, barrier protective cAMP, the toxic bacterial soluble AC, ExoY, generates, barrier disruptive cAMP and increases permeability. Increased permeability contributes to the mortality and morbidity of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS). Thus, understanding how NBCs regulate AC10 activity and how endogenously generated cytosolic cAMP regulates pulmonary endothelial barrier integrity to exacerbate ALI and ARDS is of clinical interest. Specifically, in vitro, in situ and i vivo experimental models will be used to determine whether Na+-bicarbonate cotransporters and AC10 contribute to bicarbonate influx and generation of cycotsolic cAMP signals in pulmonary endothelial cells derived from either conduit arteries or the gas exchange segment of the vasculature, the septal capillaries (Specific Aim 1). We will determine whether the NBC, NBCn2, is inhibited by juxtamembrane cAMP signals (Specific Aim 2) and whether bicarbonate transport through NBCn2 to activate AC10 is required disrupt microtubule architecture and disrupt the endothelial barrier (Specific Aim 3). Techniques to be used include: immunohistochemistry and fractionation studies to determine NBC expression between different vascular beds; pH and sodium measurements to determine NBC activity; both cell-free and in-tact cell measurements of cAMP using cAMP turnover and radioimmunoassay in addition to spatial and temporal FRET-cAMP measurements using hyperspectral image analysis; immunoprecipitation assays and site directed mutagenesis to identify phosphorylation sites on NBCn2; Western analysis with phospho-specific antibody to detect changes in tau phosphorylation; detection by detection of free versus polymerized tubulin to identify changes in microtubule architecture; video microscopy, permeability and resistance measurements to detect changes in endothelial barrier integrity in vitro; and the isolated perfused lung model to measure the filtration coefficient (Kf), lung wet/dry ratio and alveolar fluid volume fraction in stu. Further, we will perform additional isolated lung studies following intraperitoneal LPS administration to induce ALI. These experimental technique and various experiment groups will be used to test our hypothesis.
The goal of the proposed research is to determine how bicarbonate and cAMP signals contirubte to pulmonary endothelial barrier disruption during acute lung injury (ALI). This project is of clinical significance because blood bicarbonate can become elevated during respiratory diseases, is in direct contact with the pulmonary endothelial cells and can activate soluble adenylyl cyclase isoform 10 (AC10) to generate barrier disruptive cAMP signals. Thus, a greater understanding of bicarbonate regulation of the pulmonary endothelial barrier will facilitate the development of novel and beneficial therapeutic interventions.
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| Nickols, Jordan; Obiako, Boniface; Ramila, K C et al. (2015) Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10. Am J Physiol Lung Cell Mol Physiol 309:L1430-7 |
| Morrow, K A; Seifert, R; Kaever, V et al. (2015) Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection. Am J Physiol Lung Cell Mol Physiol 309:L1199-207 |
