(Verbatim from the application): H/Reox and inflammatory mediators like histamine generate reactive oxygen species (ROS) in the vascular endothelium, increase endothelial adhesivity for polymorphonuclear leukocytes (PMNs), and stimulate endothelial intracellular calcium ([Ca2+]i) oscillations. The studies in this amended proposal test the hypothesis that ROS affect endothelial Ca2+ signaling which, in turn, regulates the expression of proinflammatory genes. Recent evidence shows that the frequency, rather than the amplitude, of [Ca2+]i oscillations regulates the expression of proinflammatory genes in certain cell types. The applicant's laboratory has now shown that [Ca2+]i oscillation frequency regulates the activity of the transcription factor NF- B in human aortic endothelial cells (HAEC) stimulated by histamine. This amended proposal will examine the causes and consequences of histamine and oxidant-stimulated [Ca2+]i oscillations in cultured HAEC and in situ in an isolated, perfused rat lung model. Cell signaling pathways leading to [Ca2+], oscillations will be characterized in HAEC using the Ca2+-sensitive fluorescent probe indo 1 and the redox sensitivity of intracellular Ca2+ stores will be examined using the low-affinity Ca2+-sensitive probe Mag-indo 1 in permeabilized HAEC. Whether [Ca2+]i oscillation frequency during agonist stimulation and oxidant stress regulates the activity of specific nuclear transcription factors and the surface expression of specific cellular adhesion molecules in HAEC will be examined using reporter gene studies, electropheretic mobility shift assays (EMSA), and flow cytometry. The relationship between intracellular pH (pHi) and [Ca2+]i oscillation frequency will be determined, since we have shown that oxidant stress (i.e., H2O2) inhibits the Na+/H+ exchanger (NHE) and decreases pHi in HAEC by activating the DNA repair enzyme poly (ADP-ribose) polymerase (PARP). The hypothesis that PARP activation and pHi modulate [Ca2+]i oscillation frequency will be specifically examined in endothelial cells cultured from mice genetically deficient in PARP. The proposed studies could lead to novel methods of regulating proinflammatory gene expression by specifically targeting upstream signaling mechanisms in the endothelium.
