Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene.
Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Bevers
E M
EM
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Verhallen
P F
PF
Visser
A J
AJ
Comfurius
P
P
Zwaal
R F
RF
D001692:Biological Transport; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004161:Diphenylhexatriene / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism*; D008560:Membrane Fluidity; D011090:Polyenes; D013917:Thrombin / Q000494:pharmacology
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D011090:Polyenes; D004161:Diphenylhexatriene; D015759:Ionomycin; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D013917:Thrombin