Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.
Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.
Ehringer
W D
WD
Center for Applied Microcirculatory Research, University of Louisville, School of Medicine, KY 40292, USA.
Niu
W
W
Chiang
B
B
Wang
O L
OL
Gordon
L
L
Chien
S
S
D015687:Cell Hypoxia; D002462:Cell Membrane; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004058:Diffusion; D004527:Egg Proteins / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers; D008081:Liposomes / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines; D014471:Umbilical Veins
D004527:Egg Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005635:Fructosediphosphates; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; C024098:6-carboxyfluorescein; C029063:fructose-1,6-diphosphate