NUCLEAR ARCHITECTURE AND CALCIUM TRANSIENTS

Inflammation is a vascular response, which causes seepage of plasma fluids
and proteins into the interstitium, and consequently edema. The key
cellular event in this response is the formation of gaps between adjoined
endothelial cells. The long-term objective of this study is to understand
how endothelial cells change shape to form gaps during inflammation. Thrombin and bradykinin are two mediators known to trigger the vascular
inflammatory response. These appear to form different types of endothelial
gaps. Thrombin forms large holes in the endothelium, whereas bradykinin
initiates formation of small, transient gaps. The current hypothesis to
explain endothelial gap formation is that mediators increase intracellular
free calcium concentration ([Ca2+]i), which then alters the cytoskeleton,
thereby forming gaps. However, thrombin elicits only a modest increase in
[Ca2+]i in comparison to the steep rise in [Ca2+]i after endothelial cells
are exposed to bradykinin. Furthermore, thrombin produces changes in
[Ca2+]i, which are diffusely seen throughout the cell, in contrast to a
very distinct calcium signal, which is localized to the nucleus, after
bradykinin treatment. Since the nucleus is known to contain a highly
organized, filamentous matrix and has attachments to the cytoskeleton, it
is possible that the rise in nuclear Ca2+ serves as a signal for
architectural changes within the nucleus, which in turn alter endothelial
cell shape. The purpose of this study is to investigate nuclear
architecture and Ca2+ in microvascular endothelial cells (MEC) from bovine
lung. The plan is to: Aim 1: Examine nuclear architecture in cultured MECs after exposure to
either thrombin or bradykinin, which elicit very different spatial Ca2+
transients. Aim 2: Manipulate Ca2+ and study nuclear structure in nuclei isolated from
MECs. The nucleus will be examined using: freeze-fracture to expose the nuclear
envelope, detergent extraction with platinum-carbon replication to view
cytoskeletal attachments to the nucleus, and extraction procedures with
embedment-free thin-sections to reveal the nuclear matrix. The
spatiotemporal alterations of [Ca2+]i in individual MECs and isolated
nuclei will be measured using differential interference contrast (DIC) and
Fura-2 fluorescence digital imaging.