Function of intracellular [Ca2+]i in exocytosis and transbilayer movement in human platelets surface-labeled with the fluorescent probe 1-(4-trimethylammonio)phenyl)-6-phenyl-1,3,5-hexatriene.
Ellipsometry indicated that 1-(4-(trimethylammonio)phenyl-6-phenylhexa-1,3,5-triene (TMA-DPH) bound to platelets in a reversible and saturable way. Accordingly, the fluorescence intensity (F) of a suspension of TMA-DPH-labeled platelets was described as a quantity, determined by the amount of TMA-DPH bound to the platelet surface. Most platelet activators elevated F to a degree that correlate well with the secretion of serotonin evoked by these activators. The increase in F levels reflected the increase in outer membrane surface area following exocytosis. However, activators that evoked prolonged (> 2.5 min) and strong (> 600 nM) elevations of cytosolic [Ca2+]i increased F to levels that were much higher than expected from the maximal increase in surface area due to exocytosis. This high increase in F was caused by inward transbilayer movement of TMA-DPH over the plasma membrane and the subsequent labeling of cytosolic membrane sides. The kinetics of exocytosis and changes in cytosolic [Ca2+]i were studied by stopped-flow mixing of platelets with agonist. Thrombin-induced exocytosis had a delay of only 3 s, which was shortened when external CaCl2 or ADP was present. This correlated well with a faster rise in [Ca2+]i in the presence of CaCl2 or ADP, indicating that exocytosis was linked in time to elevation of [Ca2+]i. By itself, ADP was unable to evoke exocytosis and it elicited a [Ca2+]i transient of much shorter duration than thrombin, but with similar maximum. We concluded that both exocytosis and transbilayer movement were associated with elevation of [Ca2+]i: exocytosis required a moderate, relatively prolonged rise and transbilayer movement was accompanied by a stronger rise of even longer duration. Influx of external Ca2+ was essential for transbilayer movement, but not for exocytosis.
Heemskerk
J W
JW
Department of Biochemistry/Human Biology, University of Limburg, Maastricht The Netherlands.
Feijge
M A
MA
Andree
H A
HA
Sage
S O
SO
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004161:Diphenylhexatriene / Q000031:analogs & derivatives* / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology
D005456:Fluorescent Dyes; D013729:Terpenes; D004161:Diphenylhexatriene; D000244:Adenosine Diphosphate; D019284:Thapsigargin; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D013917:Thrombin; D002122:Calcium Chloride; D002118:Calcium; D016257:Fura-2