Degradation and intracellular accumulation of a residualizing hyaluronan derivative by liver endothelial cells.
The release and intracellular accumulation of 125I-hyaluronan degradation products was studied in cultured liver endothelial cells with hyaluronan oligosaccharides (relative molecular mass = approximately 44,000) uniquely modified and radiolabeled at the terminal reducing sugar. Two methods were combined to measure 125I-hyaluronan degradation by liver endothelial cells. (a) Cetylpyridinium chloride precipitation of hyaluronan oligosaccharides was used as a rapid, convenient assay to monitor the appearance of hyaluronan degradation products. Hyaluronan oligosaccharides less than 54 to 60 monosaccharides in length were not precipitated with cetylpyridinium chloride and thus were assessed as degraded. (b) Gel filtration chromatography was used to estimate the size range of oligosaccharides produced by liver endothelial cells. After internalization of 125I-hyaluronan, liver endothelial cells released radioactive degradation products into the culture media after a lag period of 2.5 to 3.0 hr. The intracellular accumulation of degraded 125I-hyaluronan was linear for at least 2 hr even though no degradation products were released. The long lag before release of degraded 125I-hyaluronan is likely caused by the modified chemical structure at the reducing end of the hyaluronan derivative; the derivative acts like a residualizing label. After this lag the release of degraded 125I-hyaluronan proceeded linearly for up to 12 hr. The extracellular 125I-hyaluronan degradation products eluted with a distribution coefficient of 1.3 on a gel filtration column. The major intracellular 125I-labeled degradation product showed the same retardation (distribution coefficient = 1.3). This retention may be caused by the hydrophobic aromatic and alkyl modifications to the former reducing sugar, also characteristics of a residualizing label. In addition, at least two larger minor intermediates were observed intracellularly. The rate of intracellular 125I-hyaluronan degradation was dependent on hyaluronan concentration and reached a maximal rate (159 molecules/cell/sec) at 2 x 10(-7) mol/L. This was about half the maximal rate of endocytosis (285 molecules/cell/sec) at a hyaluronan concentration of 1.3 x 10(-7) mol/L. The apparent ligand concentration that gives half-maximal responses for endocytosis and intracellular degradation was 0.6 x 10(-7) and 1.0 x 10(-7) mol/L, respectively.
McGary
C T
CT
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647.
Yannariello-Brown
J
J
Kim
D W
DW
Stinson
T C
TC
Weigel
P H
PH
D000818:Animals; D002478:Cells, Cultured; D002594:Cetylpyridinium; D011232:Chemical Precipitation; D004705:Endocytosis; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D051381:Rats
D009844:Oligosaccharides; D006820:Hyaluronic Acid; D002594:Cetylpyridinium