Pulmonary microvascular endothelial cells (PMVECs) possess strongly adherent cell-cell junctionsthat are necessary to limit fluid, solute and macromolecule permeability into interstitial and alveolarcompartments, which is important for efficient gas exchange. PMVEC junction strength is dynamicallyadjusted by intracellular cAMP concentrations. The type 6 adenylyl cyclase (AC6) synthesizes cAMP at thecell membrane. cAMP signaling is targeted to physiologically relevant effector molecules by type 4phosphodiesterases (PDE4), specifically the -D4 isoform, which is membrane-localized by spectrin. Themembrane-localized cAMP pool strengthens PMVEC barrier function. In contrast, Pseudomonas aeruginosaintroduces a soluble adenylyl cyclase toxin, ExoY, into PMVECs that generates a cytosolic cAMP pool.cAMP synthesis within the cytosol disrupts, rather than strengthens, the PMVEC barrier. To determinewhether membrane or cytosolic AC activity dominates in control of endothelial cell barrier function, weutilized a chimeric mammalian soluble AC enzyme that could be activated by forskolin. Simultaneousstimulation of membrane and cytosolic AC activity by forskolin disrupts, rather than strengthens, the PMVECbarrier, indicating soluble AC activity dominantly controls barrier strength. Preliminary data suggest solubleACs associate with the centrosome and its associated microtubules, and may therefore reorganizemicrotubule architecture necessary to induce PMVEC gaps. Thus, this proposal tests the overall hypothesisthat membrane-localized ACs produce a cAMP pool that strengthens, whereas cytosolic ACs produce acAMP pool that disrupts, the PMVEC barrier.
Specific Aims test the related Hypotheses that: [1] AC6generates a membrane cAMP pool that is maintained by a spectrin and PDE4(D4) interaction; [2] SolubleACs generate a cytosolic cAMP pool that controls microtubule organization; and, [3] cAMP that accesses thecytosolic compartment disassembles microtubules and disrupts the endothelial cell barrier. Completion ofthis work will contribute to our understanding of how cAMP acts to control PMVEC barrier strength, and willseek to resolve pathogenic mechanisms of bacteria like Pseudomonas aeruginosa, which utilize adenylylcyclase toxins to disrupt the endothelial cell barrier and increase permeability.
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