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Rate, extent and concentration dependence of histamine‐evoked Weibel–Palade body exocytosis determined from individual fusion events in human endothelial cells
Author(s) -
Erent M.,
Meli A.,
Moisoi N.,
Babich V.,
Hannah M. J.,
Skehel P.,
Knipe L.,
Zupančič G.,
Ogden D.,
Carter T.
Publication year - 2007
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2007.132993
Subject(s) - exocytosis , histamine , extracellular , chemistry , biophysics , intracellular , secretion , fluorescence , microbiology and biotechnology , biochemistry , biology , endocrinology , physics , quantum mechanics
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 μ m histamine and 1.57 ± 0.21 s ( n = 15 cells) at 100 μ m 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 μ m and 3.66 ± 0.45 WPB s −1 at 100 μ m 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 Ca 2+ indicating that the initial burst of secretion is driven by Ca 2+ release from internal stores, but sustained exocytosis required external Ca 2+ . Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μ m ), 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 .