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J Physiol Volume 544, Number 3, 741-755, November 1, 2002 DOI: 10.1113/jphysiol.2002.027490
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Journal of Physiology (2002), 544.3, pp. 741-755
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.027490

Differential exocytosis from human endothelial cells evoked by high intracellular Ca2+ concentration

G. Zupani*, D. Ogden, C. J. Magnus, C. Wheeler-Jones† and T. D. Carter‡

National Institute for Medical Research, Mill Hill, London NW7 1AA, UK, *University of Ljubljana, Department of Biology, Vec na Pot 111, 1001 Ljubljana, POB 2995, Slovenia, †Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK and ‡Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK

Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high-resolution membrane capacitance (Cm) measurements. The total whole-cell Cm and the amplitudes and times of discrete femtoFarad (fF)-sized Cm steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca2+]i was elevated by intracellular photolysis of calcium-DM-nitrophen to evoke secretion and monitored with the low-affinity Ca2+ indicator furaptra. Sustained elevation of [Ca2+]i to > 20 µM evoked large, slow increases in Cm of up to 5 pF in 1-2 min. Exocytotic and endocytotic steps of amplitude 0.5-110 fF were resolved, and accounted on average for ~33 % of the total Cm change. A prominent component of Cm steps of 2.5-9.0 fF was seen and could be attributed to exocytosis of von-Willebrand-factor-containing Weibel-Palade bodies (WPb), based on the near-identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5-9.0 fF Cm steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca2+]i elevation, whereas total Cm increased immediately due to the secretion of small, non-WPb granules. The results show that following a large increase of [Ca2+]i, corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9-110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.



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