Most existing knowledge about [Ca(2+)](i) signaling in vascular endothelium has been based on studies using endothelial cells cultured in vitro. To examine how endothelial cells behave in situ, we have developed a method to monitor single-cell [Ca(2+)](i) from Fura-2-loaded rat aortic segments. Fluorescence ratio images from large numbers of endothelial cells were acquired by using a flow chamber mounted on a dual-wavelength fluorescence microscope. Our results showed that either acetylcholine or histamine reversibly activated the vascular endothelium by eliciting M(3) or H(1) receptor-mediated [Ca(2+)](i) increases, respectively. The acetylcholine-evoked endothelial [Ca(2+)](i) elevation at the branch site (intercostal orifice) was much more pronounced than that at the non-branch area. However, endothelium at the branch site was relatively insensitive to histamine. Both acetylcholine-sensitive and histamine-sensitive endothelial cells were arranged in belts aligned along flow lines and were intercalated with each other. Data analyzed from 400 endothelial cells located at the non-branch site showed drastically heterogeneous [Ca(2+)](i) responses to a fixed concentration of either acetylcholine or histamine, differing by two orders of magnitude in individual cells. As a conclusion, vascular endothelial cells appear to have their own characteristic [Ca(2+)](i) 'fingerprint' to various agonists and they may function coordinately in situ.

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca(2+) signaling, we examined intracellular Ca(2+) concentration ([Ca(2+)](i)) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca(2+)](i) responses; 2) ACh induced heterogeneous [Ca(2+)](i) elevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca(2+)](i) elevation was inhibited by the Ca(2+) influx blocker SKF-96365, by a Ca(2+)-free buffer, or by high concentrations of extracellular K(+). We conclude that chronic exercise increases ACh-induced [Ca(2+)](i) elevation in rat aortic endothelium in situ, possibly by facilitating Ca(2+) influx.