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.

This study explores the neuroprotective action of tumor necrosis factor-a (TNF-a) induced during physical exercise, which, consequently, reduces matrix metalloproteinase-9 (MMP-9) activity and ameliorates blood-brain barrier (BBB) dysfunction in association with extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Adult male Sprague-Dawley rats were subjected to exercise on a treadmill for 3 weeks. A 2-h middle cerebral artery occlusion and reperfusion was administered to exercised and nonexercised animals to induce stroke. Exercised ischemic rats were subjected to TNF-a inhibition and ERK1/2 by TNF-a antibody or UO126. Nissl staining of coronal sections revealed the infarct volume. Evans blue extravasation and water content evaluated BBB function. Western blot was performed to analyze protein expression of TNF-a, ERK1/2, phosphorylated ERK1/2, the basal laminar protein collagen IV, and MMP-9. The activity of MMP-9 was determined by gelatin zymography. Tumor necrosis factor-a expression and ERK1/2 phosphorylation were upregulated during exercise. Infarct volume, brain edema, and Evans blue extravasation all significantly decreased in exercised ischemic rats. Collagen IV production increased in exercised rats and remained high after stroke, whereas MMP-9 protein level and activity decreased. These results were negated and returned toward nonexercised values once TNF-a or ERK1/2 was blocked. We concluded that preischemic, exercise-induced TNF-a markedly decreases BBB dysfunction by using the ERK1/2 pathway.

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