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CARDIOVASCULAR |
i3
1 Medical Research Councils National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
2 Department of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, UK
3 University of Ljubljana, Vena pot 111, PO Box 2995, 1001 Ljubljana, Slovenia
The rate, concentration dependence and extent of histamine-evoked Weibel–Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 ± 0.26 (S.D., n
= 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s (n
= 9 cells) at 0.3 µM histamine and 1.57 ± 0.21 s (n
= 15 cells) at 100 µM histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s–1 (n
= 9) at 0.3 µM and 3.66 ± 0.45 WPB s–1 at 100 µM histamine (n
= 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 µM), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n
= 6), and a peak fusion rate of 
10 WPB s–1.
(Received 21 March 2007;
accepted after revision 29 May 2007;
first published online 31 May 2007)
Corresponding author T. Carter: Medical Research Councils National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. Email: tcarter{at}nimr.mrc.ac.uk
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