Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells.
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 microM histamine and 1.57+/-0.21 s (n=15 cells) at 100 microM 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 microM and 3.66+/-0.45 WPB s(-1) at 100 microM 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 microM), 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 approximately 10 WPB s(-1).
Erent
M
M
Medical Research Councils National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Meli
A
A
Moisoi
N
N
Babich
V
V
Hannah
M J
MJ
Skehel
P
P
Knipe
L
L
Zupancic
G
G
Ogden
D
D
Carter
T
T
D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006632:Histamine / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D013997:Time Factors; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism
D007476:Ionophores; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins; C067297:8-aminohexylamino cAMP; D015759:Ionomycin; D006632:Histamine; D000242:Cyclic AMP; D002118:Calcium