Endothelial cells line all of our blood vessels. They monitor and respond to
signals generated during injury, infection and disease by releasing a wide range of
molecules that regulate blood flow, coagulation, inflammatory responses and
vessel growth. Protein mediators are released by exocytosis of intracellular
organelles, and a major trigger for this type of secretion is an increase in
intracellular free calcium ion concentration ([Ca2+]i). Mitochondria are thought to
influence Ca2+ homeostasis through local Ca2+ buffering. Due to a lack of
sensitive and time-resolved assays for endothelial exocytosis little is known about
the precise relationship between Ca2+-signalling and exocytosis, and the influence
of Ca2+ buffering by mitochondria.
Using fluorescence and biochemical techniques I have investigated the
relationship between secretagogue-evoked Ca2+-signalling and the influence of
mitochondrial function on the exocytosis of two distinct organelle populations in
cultures of Human Umbilical Vein Endothelial Cells (HUVEC); 1) the Weibel-Palade body (WPB) the main storage organelle for pro-coaguland and
inflammatory mediators, and 2) the non-WPB, a non-stored and morphologically distinct organelle that can contain a range of inflammatory and anti-coagulant
molecules.
These two distinct organelle populations were labeled for fluorescence
microscopy by targeted expression of chimeras of green (EGFP) or red (mRFP)
fluorescent proteins in living HUVEC. Exocytosis was evoked by both
physiological and pharmacological secretogogues that increase [Ca2+]i. The times
of exocytosis of individual organelles were monitored by flashes of light from
granule EGFP, produced by pH changes within the organelle upon fusion. In the
same experiments, [Ca2+]i and intra-mitochondrial Ca2+ concentration ([Ca2+]m)
were monitored using fluorescent Ca2+-indicators.
The data obtained has defined more precisely the relationship between
agonist-evoked changes in [Ca2+]i and secretory vesicle exocytosis in HUVEC.
These studies will contribute to a better understanding of the processes that
regulate secretion of biomolecules from the endothelium
