Abstract
The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.
Highlights
Theprincipal mechanism of cellularuptake of throughthemembrane (Davison and Danielli, 1943)
Inbenzo(a)pyrene and otherpolycyclic aromatic hydro- creased hydrophobicity of molecules has been correlated with carbons (PAH) from lipoproteins into cells is sponta- more rapid rates of uptake
Solutions-Lipoproteins were prepared by ultracentrifugal flotation (Have1et al., 1955) to obtain VLDL ( p < 1.006), LDL
Summary
Passive uptake of extremely phatidylcholine vesicles, even though rate constants hydrophobic fatty acids by cells has been studied (Sallee for benzo(a)pyrene transfer from these donvoerhicles and Dietschy, 1973; Sherrill andDietschy, 1975). Cule from a hydrophobic environment for dissolution in the [G-3H]benzo(a)pyrenewas added to 2 ml of RPMI 1640 medium or aqueous phase is the rate-limiting step, whereas diffusion of standard buffer containing 2 X lo6P388D1macrophages. To initate a kinetic experiment, medium containing benzo(a)pyrene-labeled LDL or HDL was added to cells on the stage through ports in the chamber lid. P388DI macrophages in dishes were suspended by rinsing with cold medium and collected by centrifugation These cultures have been previously shown not to metabolize benzo(a)pyrene during these experiments
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