Nitric Oxide and Microvessel Permeability In Vivo

Project: Research project

Project Details

Description

Nitric oxide (NO) is an important signaling molecule involved in the regulation of many physiological and
pathological functions of the vascular endothelium. The objectives of this proposal are to investigate the
mechanisms regulating endothelial NO synthase (eNOS) activity in intact microvessels under inflammatory
conditions and their direct relationship with microvessel permeability. The hypothesis to be tested is that
Ca2+/calmodulin (CaM) and heat shock protein 90 (hsp90) play antagonistic roles to caveolin-1 in the
regulation of eNOS activity and microvessel permeability through competing protein-protein interactions in
intact microvessels, and agonist-stimulated NO production is critical for increases in permeability under
inflammatory conditions. Individually perfused intact venular microvessels in rat mesentery will be used to
perform the proposed studies. The unique advantage of using single vessel perfusion technique over other
whole animal approaches for this specific study is that it allows the NO effect on blood cell/endothelium
interactions and on the changes in hemodynamics in the vasculature to be distinguished from its role in the
regulation of microvessel permeability. Agonist-stimulated NO production will be quantitatively measured at
levels of individual endothelial cells in intact microvessels with temporal and spatial resolution using
fluorescence imaging. The role of Ca2+/CaM in the regulation of eNOS activity will be investigated by
measuring corresponding changes in NO production when agonist-induced Ca2+ influx is modified following
changes in electrochemical driving force for Ca2+ entry in endothelial cells. Our newly developed methods
also allow a targeted regulatory protein for eNOS such as caveolin-1 or hsp90 to be internalized or
genetically expressed in endothelial cel|s that form intact microvessels. In this proposal we combined our
newly developed molecular approaches with our previously established techniques to investigate the
mechanisms regulating eNOS activity in intact microvessels. Because of the nonuniformity nature of leaky
site formation along the microvessel walls during acute inflammation, the spatial heterogeneity correlation
between endothelial [Ca2+]i and NO production will be quantitatively evaluated at cellular levels in intact
microvessels. The proposed research will provide new information that bridges the studies using whole
animal, organ, or vascular beds and studies with purified proteins or endothelial cells in culture.
StatusFinished
Effective start/end date1/19/0712/31/13

Funding

  • National Heart, Lung, and Blood Institute: $329,625.00
  • National Heart, Lung, and Blood Institute: $329,625.00
  • National Heart, Lung, and Blood Institute: $329,625.00
  • National Heart, Lung, and Blood Institute: $329,625.00
  • National Heart, Lung, and Blood Institute: $329,625.00

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