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.
| Merkle, C J; Schuler, L A; Schaeffer Jr, R C et al. (2000) Structural and functional effects of high prolactin levels on injured endothelial cells: evidence for an endothelial prolactin receptor. Endocrine 13:37-46 |
| Yamamoto, N S; Merkle, C J; Kraus-Friedmann, N (1999) Disruption of filamentous actin diminishes hormonally evoked Ca2+ responses in rat liver. Metabolism 48:1241-7 |
